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Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in <t>GATA4-expressing</t> (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. <t>RT-PCR</t> was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI ( ROSA26 lnlG4/+ Villin-Cre ) and control ( ROSA26 lnlG4/+ ) mice and in jejunal epithelial cells from Gata4 cKO ( Gata4 loxP/loxP Villin-Cre ) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold . Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. * P ≤ .05. ** P ≤ .01.
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1) Product Images from "GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine"

Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

Journal: Cellular and Molecular Gastroenterology and Hepatology

doi: 10.1016/j.jcmgh.2016.12.009

Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in GATA4-expressing (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. RT-PCR was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI ( ROSA26 lnlG4/+ Villin-Cre ) and control ( ROSA26 lnlG4/+ ) mice and in jejunal epithelial cells from Gata4 cKO ( Gata4 loxP/loxP Villin-Cre ) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold . Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. * P ≤ .05. ** P ≤ .01.
Figure Legend Snippet: Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in GATA4-expressing (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. RT-PCR was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI ( ROSA26 lnlG4/+ Villin-Cre ) and control ( ROSA26 lnlG4/+ ) mice and in jejunal epithelial cells from Gata4 cKO ( Gata4 loxP/loxP Villin-Cre ) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold . Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. * P ≤ .05. ** P ≤ .01.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Mouse Assay

Duodenal and jejunal epithelial cells in Gata4 cKI mice express normal levels of GATA4. ( A ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in duodenal and jejunal epithelium of Gata4 cKI mice at similar staining intensity compared with controls. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( B ) qRT-PCR showed that Gata4 mRNA was unchanged in epithelial cells of the duodenum and jejunum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with control mice ( ROSA26 lnlG4/+ ) (n = 3 per genotype; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test. ( C ) Immunoblot analysis of nuclear extracts from duodenal and jejunal epithelial cells of control and Gata4 cKI mice was used to quantify GATA4 protein (n = 3 per genotype). To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression was unchanged in duodenum and jejunum of Gata4 cKI animals compared with control. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test.
Figure Legend Snippet: Duodenal and jejunal epithelial cells in Gata4 cKI mice express normal levels of GATA4. ( A ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in duodenal and jejunal epithelium of Gata4 cKI mice at similar staining intensity compared with controls. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( B ) qRT-PCR showed that Gata4 mRNA was unchanged in epithelial cells of the duodenum and jejunum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with control mice ( ROSA26 lnlG4/+ ) (n = 3 per genotype; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test. ( C ) Immunoblot analysis of nuclear extracts from duodenal and jejunal epithelial cells of control and Gata4 cKI mice was used to quantify GATA4 protein (n = 3 per genotype). To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression was unchanged in duodenum and jejunum of Gata4 cKI animals compared with control. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test.

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Quantitative RT-PCR, Expressing, Software, Binding Assay, Molecular Weight, Marker

Enterohepatic signaling is altered in animals expressing GATA4 in the ileum. ( A ) Immunohistochemistry for SLC10A2 (brown stain) shows SLC10A2 protein lining the brush border of control ileum (n = 7, upper panel). In contrast, SLC10A2 staining was faint to nearly absent along the brush border of ileum from Gata4 cKI mice (n = 14, 7 of 14 faint SLC10A2 staining, middle panel , and 7 of 14 low to no SLC10A2 staining, lower panel ). Immunohistochemistry from 2 independent Gata4 cKI mice is shown as representative of the 2 types of SLC10A2 staining observed. ( B ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of SLC10A2 protein. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. SLC10A2 protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. SLC10A2 expression in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 9% of the level observed in control ileum ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Arrowhead indicates the SLC10A2 band measured. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( C ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of OSTα and OSTβ proteins. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. OSTα and OSTβ protein levels were normalized to GAPDH levels. Expression of OSTα and OSTβ proteins in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 26% and 19% of the level observed in control ileum ( ROSA26 lnlG4/+ ), respectively (n = 3 animals per genotype). Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) qRT-PCR shows increased Cyp7a1 expression in liver from Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) compared with control mice ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars represent SEM. * P ≤ .05.
Figure Legend Snippet: Enterohepatic signaling is altered in animals expressing GATA4 in the ileum. ( A ) Immunohistochemistry for SLC10A2 (brown stain) shows SLC10A2 protein lining the brush border of control ileum (n = 7, upper panel). In contrast, SLC10A2 staining was faint to nearly absent along the brush border of ileum from Gata4 cKI mice (n = 14, 7 of 14 faint SLC10A2 staining, middle panel , and 7 of 14 low to no SLC10A2 staining, lower panel ). Immunohistochemistry from 2 independent Gata4 cKI mice is shown as representative of the 2 types of SLC10A2 staining observed. ( B ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of SLC10A2 protein. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. SLC10A2 protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. SLC10A2 expression in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 9% of the level observed in control ileum ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Arrowhead indicates the SLC10A2 band measured. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( C ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of OSTα and OSTβ proteins. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. OSTα and OSTβ protein levels were normalized to GAPDH levels. Expression of OSTα and OSTβ proteins in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 26% and 19% of the level observed in control ileum ( ROSA26 lnlG4/+ ), respectively (n = 3 animals per genotype). Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) qRT-PCR shows increased Cyp7a1 expression in liver from Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) compared with control mice ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars represent SEM. * P ≤ .05.

Techniques Used: Expressing, Immunohistochemistry, Staining, Mouse Assay, Software, Molecular Weight, Marker, Quantitative RT-PCR

Expression of jejunal and duodenal transcripts is unchanged in Gata4 cKI animals. ( A ) qRT-PCR was used to determine transcript abundance of the 10 jejunal-enriched transcripts, identified as having enriched GATA4 binding by bio-ChIP–PCR ( Figure 7 ) in jejunal epithelial cells from control and Gata4 cKI mice. ( B ) qRT-PCR was used to determine transcript abundance of 4 duodenal transcripts in duodenal epithelial cells from control and Gata4 cKI mice. ( A and B ) Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments (n = 3 per genotype). Error bars represent SEM. P values were determined by 2-sample Student t test.
Figure Legend Snippet: Expression of jejunal and duodenal transcripts is unchanged in Gata4 cKI animals. ( A ) qRT-PCR was used to determine transcript abundance of the 10 jejunal-enriched transcripts, identified as having enriched GATA4 binding by bio-ChIP–PCR ( Figure 7 ) in jejunal epithelial cells from control and Gata4 cKI mice. ( B ) qRT-PCR was used to determine transcript abundance of 4 duodenal transcripts in duodenal epithelial cells from control and Gata4 cKI mice. ( A and B ) Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments (n = 3 per genotype). Error bars represent SEM. P values were determined by 2-sample Student t test.

Techniques Used: Expressing, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Mouse Assay

Representative autoradiographs of bio-ChIP–PCR. Bio-ChIP–PCR was used to evaluate GATA4 occupancy at predicted binding sites in the 26 high-confidence direct targets we identified and in 7 negative controls ( Alb, Cdk4, Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ). GATA4 occupied chromatin was isolated by performing streptavidin pull-down with chromatin from jejunal epithelial cells of GATA4-FlagBio/BirA or GATA4-WT/BirA mice. As representative data, PCR with chromatin from 2 mice per genotype is shown here. Input PCR confirmed that equivalent chromatin amounts were used in pull-downs. In all, 6 mice per genotype were assayed by bio-ChIP–PCR.
Figure Legend Snippet: Representative autoradiographs of bio-ChIP–PCR. Bio-ChIP–PCR was used to evaluate GATA4 occupancy at predicted binding sites in the 26 high-confidence direct targets we identified and in 7 negative controls ( Alb, Cdk4, Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ). GATA4 occupied chromatin was isolated by performing streptavidin pull-down with chromatin from jejunal epithelial cells of GATA4-FlagBio/BirA or GATA4-WT/BirA mice. As representative data, PCR with chromatin from 2 mice per genotype is shown here. Input PCR confirmed that equivalent chromatin amounts were used in pull-downs. In all, 6 mice per genotype were assayed by bio-ChIP–PCR.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Isolation, Mouse Assay

Gata4 conditional knock-in mice express GATA4 in the ileum. ( A ) Schematic illustrating the strategy used to generate a conditional Gata4 knock-in mouse line. The coding sequence of the mouse Gata4 gene was amplified by PCR and inserted into XhoI/SacI sites in the multiple cloning site (MCS) of pBig-T to generate pBigT- Gata4 . The targeting cassette consisting of an adenoviral splice acceptor (SA), a loxP flanked phosphoglycerate kinase (PGK) promoter-neomycin resistance gene (Neo) and 3×SV40 polyadenylation sequence (pA) sequence ( loxP -PGK-Neo-3×SV40pA- loxP , LNL), the Gata4 coding sequence, and a bovine growth hormone polyadenylation (pA) sequence was excised from pBigT- Gata4 with PacI/AscI and inserted into the PacI/AscI sites in pROSA26PA to create pROSA26PA- Gata4 . Homologous recombination between pROSA26PA- Gata4 and the endogenous ROSA26 locus in mouse R1 embryonic stem cells yielded the targeted locus Gt(ROSA)26Sor tm1(Gata4)Bat , designated ROSA26 lnlG4 . After Cre recombination to excise the LNL cassette, Gata4 is expressed. BamHI ( B ) and EcoRV ( E ) restriction sites used for Southern blot analysis, the position of Southern blot probes, and relevant BamHI and EcoRV restriction digest fragments identified by Southern blot are shown. Arrows mark sites of genotyping primers ( Table 1 , primers). ( B ) Southern blot analysis confirmed germline transmission of the ROSA26 lnlG4 allele. Representative Southern blot analysis of EcoRV or BamHI digested genomic DNA harvested from a wild-type mouse (ROSA26 +/+ ) or a mouse heterozygous for the modified ROSA26 allele ( ROSA26 lnlG4/+ ). We observed the expected fragments representing the wild-type and modified alleles ( EcoRV digest, 11.5-kb wild-type allele and 4.0-kb modified allele; BamHI digest, 5.8-kb wild-type allele and 4.7-kb modified allele). ( C ) qRT-PCR showed that Gata4 mRNA was induced in ileum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with ileum of control mice ( ROSA26 lnlG4/+ ). Gata6 mRNA remained unchanged in the ileum of Gata4 cKI mice compared with controls (n = ileum of 5 control and 6 Gata4 cKI animals; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in ileal epithelium of Gata4 cKI mice and in the jejunal epithelium of control mice whereas GATA4 protein was absent from ileal epithelium of control mice. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( E ) Immunoblot analysis of nuclear extracts from jejunal and ileal epithelial cells of control mice and from ileal epithelial cells of Gata4 cKI mice was used to quantify GATA4 protein in ileum of Gata4 cKI mice and to compare GATA4 abundance between control jejunum and GATA4-expressing ileum. The blot shown contains nuclear protein extracts from 3 control and 3 Gata4 cKI animals and is representative of analysis of more than 24 control and Gata4 cKI animals. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression in ileum of Gata4 cKI mice was 27% the level observed in control jejunum. Molecular weight marker locations are indicated. Error bars show SEM. P values determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .001.
Figure Legend Snippet: Gata4 conditional knock-in mice express GATA4 in the ileum. ( A ) Schematic illustrating the strategy used to generate a conditional Gata4 knock-in mouse line. The coding sequence of the mouse Gata4 gene was amplified by PCR and inserted into XhoI/SacI sites in the multiple cloning site (MCS) of pBig-T to generate pBigT- Gata4 . The targeting cassette consisting of an adenoviral splice acceptor (SA), a loxP flanked phosphoglycerate kinase (PGK) promoter-neomycin resistance gene (Neo) and 3×SV40 polyadenylation sequence (pA) sequence ( loxP -PGK-Neo-3×SV40pA- loxP , LNL), the Gata4 coding sequence, and a bovine growth hormone polyadenylation (pA) sequence was excised from pBigT- Gata4 with PacI/AscI and inserted into the PacI/AscI sites in pROSA26PA to create pROSA26PA- Gata4 . Homologous recombination between pROSA26PA- Gata4 and the endogenous ROSA26 locus in mouse R1 embryonic stem cells yielded the targeted locus Gt(ROSA)26Sor tm1(Gata4)Bat , designated ROSA26 lnlG4 . After Cre recombination to excise the LNL cassette, Gata4 is expressed. BamHI ( B ) and EcoRV ( E ) restriction sites used for Southern blot analysis, the position of Southern blot probes, and relevant BamHI and EcoRV restriction digest fragments identified by Southern blot are shown. Arrows mark sites of genotyping primers ( Table 1 , primers). ( B ) Southern blot analysis confirmed germline transmission of the ROSA26 lnlG4 allele. Representative Southern blot analysis of EcoRV or BamHI digested genomic DNA harvested from a wild-type mouse (ROSA26 +/+ ) or a mouse heterozygous for the modified ROSA26 allele ( ROSA26 lnlG4/+ ). We observed the expected fragments representing the wild-type and modified alleles ( EcoRV digest, 11.5-kb wild-type allele and 4.0-kb modified allele; BamHI digest, 5.8-kb wild-type allele and 4.7-kb modified allele). ( C ) qRT-PCR showed that Gata4 mRNA was induced in ileum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with ileum of control mice ( ROSA26 lnlG4/+ ). Gata6 mRNA remained unchanged in the ileum of Gata4 cKI mice compared with controls (n = ileum of 5 control and 6 Gata4 cKI animals; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in ileal epithelium of Gata4 cKI mice and in the jejunal epithelium of control mice whereas GATA4 protein was absent from ileal epithelium of control mice. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( E ) Immunoblot analysis of nuclear extracts from jejunal and ileal epithelial cells of control mice and from ileal epithelial cells of Gata4 cKI mice was used to quantify GATA4 protein in ileum of Gata4 cKI mice and to compare GATA4 abundance between control jejunum and GATA4-expressing ileum. The blot shown contains nuclear protein extracts from 3 control and 3 Gata4 cKI animals and is representative of analysis of more than 24 control and Gata4 cKI animals. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression in ileum of Gata4 cKI mice was 27% the level observed in control jejunum. Molecular weight marker locations are indicated. Error bars show SEM. P values determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .001.

Techniques Used: Knock-In, Mouse Assay, Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, Southern Blot, Transmission Assay, Modification, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing, Software, Binding Assay, Molecular Weight, Marker

GATA4 occupies sites in jejunal- and ileal-enriched genes, suggesting GATA4 directly regulates expression of jejunal- and ileal-enriched genes in the jejunum to define jejunal enterocyte identity. Bio-ChIP–PCR showed GATA4 enrichment at GATA4 binding sites within genes expressed in jejunum ( top panel ) and within genes repressed in jejunum ( middle panel ). No GATA4 enrichment was observed at sites lacking GATA4 bio-ChIP-seq binding sites ( Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ) or in genes identified as GATA4 targets in other tissues but that are either equivalently expressed in ileum of control and Gata4 cKI mice ( Cdk4 ) or absent in ileum of control and Gata4 cKI mice ( Alb ) ( bottom panel ). Audioradiographic band intensity was measured using a Storm820 Phosphor Imager and ImageQuant software. Representative autoradiographs for each site assayed are shown in Figure 7 . Enrichment per sample was normalized to input (n = 6 Gata4 flbio/flbio ::ROSA26 BirA/BirA mice, designated GATA4-FlagBio/BirA , and 6 ROSA26 BirA/BirA mice, designated GATA4-WT/BirA ). Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .005. P values > .05 are listed on graphs. GATA4 occupancy at the binding sites in the Slc10a2 gene ( Slc10a2 _1 and Slc10a2 _2) was analyzed previously by qPCR. 38
Figure Legend Snippet: GATA4 occupies sites in jejunal- and ileal-enriched genes, suggesting GATA4 directly regulates expression of jejunal- and ileal-enriched genes in the jejunum to define jejunal enterocyte identity. Bio-ChIP–PCR showed GATA4 enrichment at GATA4 binding sites within genes expressed in jejunum ( top panel ) and within genes repressed in jejunum ( middle panel ). No GATA4 enrichment was observed at sites lacking GATA4 bio-ChIP-seq binding sites ( Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ) or in genes identified as GATA4 targets in other tissues but that are either equivalently expressed in ileum of control and Gata4 cKI mice ( Cdk4 ) or absent in ileum of control and Gata4 cKI mice ( Alb ) ( bottom panel ). Audioradiographic band intensity was measured using a Storm820 Phosphor Imager and ImageQuant software. Representative autoradiographs for each site assayed are shown in Figure 7 . Enrichment per sample was normalized to input (n = 6 Gata4 flbio/flbio ::ROSA26 BirA/BirA mice, designated GATA4-FlagBio/BirA , and 6 ROSA26 BirA/BirA mice, designated GATA4-WT/BirA ). Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .005. P values > .05 are listed on graphs. GATA4 occupancy at the binding sites in the Slc10a2 gene ( Slc10a2 _1 and Slc10a2 _2) was analyzed previously by qPCR. 38

Techniques Used: Expressing, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Mouse Assay, Software, Real-time Polymerase Chain Reaction

2) Product Images from "Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis"

Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

Journal: Journal of Clinical Investigation

doi:

Identification of WHV RNA sequences in naive and mitogen-stimulated PBMCs in offspring born to mothers convalescent from viral hepatitis. Total RNA was extracted from ( a ) unstimulated PBMCs from 4B/M offspring obtained at 18, 30, and 32 months after birth and from ( b ) unstimulated (–ConA) and Con A–stimulated (+ConA) PBMCs from 11D/F offspring at 34.5 months after birth. RNA isolated from Con A–stimulated PBMCs of a WHV-negative, healthy woodchuck (Control PBMCs + ConA) and from the spleen of a WHsAg-positive chronic WHV carrier (Positive control) was used as control. For all RT reactions, cDNA synthesis was carried out by RT (RT+) and the cDNA analyzed by nested PCR/Southern blot hybridization. Each RNA sample was subjected to the same reaction conditions in the absence of RT (RT–).
Figure Legend Snippet: Identification of WHV RNA sequences in naive and mitogen-stimulated PBMCs in offspring born to mothers convalescent from viral hepatitis. Total RNA was extracted from ( a ) unstimulated PBMCs from 4B/M offspring obtained at 18, 30, and 32 months after birth and from ( b ) unstimulated (–ConA) and Con A–stimulated (+ConA) PBMCs from 11D/F offspring at 34.5 months after birth. RNA isolated from Con A–stimulated PBMCs of a WHV-negative, healthy woodchuck (Control PBMCs + ConA) and from the spleen of a WHsAg-positive chronic WHV carrier (Positive control) was used as control. For all RT reactions, cDNA synthesis was carried out by RT (RT+) and the cDNA analyzed by nested PCR/Southern blot hybridization. Each RNA sample was subjected to the same reaction conditions in the absence of RT (RT–).

Techniques Used: Isolation, Positive Control, Nested PCR, Southern Blot, Hybridization

Detection of WHV cccDNA in selected WHV DNA–reactive liver, PBMCs, and lymphoid tissue samples from 3B/M, 4B/M, and 5C/F offspring. DNA was extracted from autopsy spleen and bone marrow of 3B/M; from liver biopsies collected at 19 and 31 months and PBMCs obtained at 34 and 41 months after birth from 4B/M; and from liver, spleen, and bone marrow collected at autopsy of 5C/F performed at 22 months of age. The PBMCs harvested from 4B/M at 41 months after birth were extensively washed and the cell surface treated with DNase and trypsin before DNA isolation. DNA samples (2 or 5 μg) were digested with mung bean endonuclease and subjected to nested PCR with primers amplifying the WHV gap-spanning region. DNA samples from a WHsAg-positive chronic carrier were included as positive controls; water instead of DNA and a mock sample extracted in parallel with test samples were used as negative controls. Positive samples showed the expected size of the amplified nucleotide fragments indicated on the left.
Figure Legend Snippet: Detection of WHV cccDNA in selected WHV DNA–reactive liver, PBMCs, and lymphoid tissue samples from 3B/M, 4B/M, and 5C/F offspring. DNA was extracted from autopsy spleen and bone marrow of 3B/M; from liver biopsies collected at 19 and 31 months and PBMCs obtained at 34 and 41 months after birth from 4B/M; and from liver, spleen, and bone marrow collected at autopsy of 5C/F performed at 22 months of age. The PBMCs harvested from 4B/M at 41 months after birth were extensively washed and the cell surface treated with DNase and trypsin before DNA isolation. DNA samples (2 or 5 μg) were digested with mung bean endonuclease and subjected to nested PCR with primers amplifying the WHV gap-spanning region. DNA samples from a WHsAg-positive chronic carrier were included as positive controls; water instead of DNA and a mock sample extracted in parallel with test samples were used as negative controls. Positive samples showed the expected size of the amplified nucleotide fragments indicated on the left.

Techniques Used: DNA Extraction, Nested PCR, Amplification

Effect of DNase digestion on WHV DNA–reactive particles circulating in an offspring born to a mother convalescent from viral hepatitis. Serum obtained at 32 months after birth from 4B/M offspring with WHV DNA expression in both liver and PBMCs; purified WHV virions; and Mnl I-digested recombinant WHV DNA were centrifuged through 15% sucrose over a 60% sucrose cushion, as described in Methods. Fifteen fractions collected from the bottom of each gradient were tested for WHV DNA by PCR, and those showing highest WHV DNA reactivity were pooled and were digested with DNase (D). Tested samples included pooled fractions 1–3 (bottom) for WHV virions, 1–5 (bottom) and 8–12 (top) for 4B/M serum, and 9–11 (top) for recombinant WHV DNA. As controls, samples of the same pooled fractions, but this time not treated with DNase (ND), were used. DNA extracted from each sample was tested for WHV S gene sequences by nested PCR and Southern blot hybridization.
Figure Legend Snippet: Effect of DNase digestion on WHV DNA–reactive particles circulating in an offspring born to a mother convalescent from viral hepatitis. Serum obtained at 32 months after birth from 4B/M offspring with WHV DNA expression in both liver and PBMCs; purified WHV virions; and Mnl I-digested recombinant WHV DNA were centrifuged through 15% sucrose over a 60% sucrose cushion, as described in Methods. Fifteen fractions collected from the bottom of each gradient were tested for WHV DNA by PCR, and those showing highest WHV DNA reactivity were pooled and were digested with DNase (D). Tested samples included pooled fractions 1–3 (bottom) for WHV virions, 1–5 (bottom) and 8–12 (top) for 4B/M serum, and 9–11 (top) for recombinant WHV DNA. As controls, samples of the same pooled fractions, but this time not treated with DNase (ND), were used. DNA extracted from each sample was tested for WHV S gene sequences by nested PCR and Southern blot hybridization.

Techniques Used: Expressing, Purification, Recombinant, Polymerase Chain Reaction, Nested PCR, Southern Blot, Hybridization

Analysis of WHV DNA expression in liver, PBMCs, and lymphoid tissues collected from 3B/M offspring. WHV gene sequences were identified by nested PCR using C and X gene–specific primers, followed by Southern blot hybridization of the amplified products to recombinant WHV DNA. Five micrograms of DNA extracted from liver samples collected at 6 months of age and at autopsy performed at 15 months after birth, and 1 μg DNA from PBMCs collected at 14.5 months of age and from spleen, lymph node, bone marrow, and skeletal muscle obtained at autopsy, were used for direct PCR amplification. Positive samples showed the expected molecular size of the amplified virus C and X gene fragments indicated on the left.
Figure Legend Snippet: Analysis of WHV DNA expression in liver, PBMCs, and lymphoid tissues collected from 3B/M offspring. WHV gene sequences were identified by nested PCR using C and X gene–specific primers, followed by Southern blot hybridization of the amplified products to recombinant WHV DNA. Five micrograms of DNA extracted from liver samples collected at 6 months of age and at autopsy performed at 15 months after birth, and 1 μg DNA from PBMCs collected at 14.5 months of age and from spleen, lymph node, bone marrow, and skeletal muscle obtained at autopsy, were used for direct PCR amplification. Positive samples showed the expected molecular size of the amplified virus C and X gene fragments indicated on the left.

Techniques Used: Expressing, Nested PCR, Southern Blot, Hybridization, Amplification, Recombinant, Polymerase Chain Reaction

WHV DNA expression in serum, liver, and lymphoid cells of 260/M woodchuck after inoculation with serum from liver WHV DNA–negative 3B/M offspring. Five micrograms of total DNA from liver biopsies collected at ∼2 months before (1) and 2 months after (2) inoculation and at autopsy (3), and 1 μg of DNA from autopsy PBMCs, isolated splenocytes, and bone marrow obtained at 3.5 months after inoculation or from 50 μL of autopsy serum, were tested for WHV DNA by nested PCR using WHV C gene–specific primers and hybridization to WHV DNA probe. DNA from serum of a WHsAg-positive chronic carrier was included as a positive control, and water instead of DNA and a mock sample extracted in parallel with test samples were used negative controls. Positive samples showed the expected 428-bp nucleotide fragment noted by arrowhead.
Figure Legend Snippet: WHV DNA expression in serum, liver, and lymphoid cells of 260/M woodchuck after inoculation with serum from liver WHV DNA–negative 3B/M offspring. Five micrograms of total DNA from liver biopsies collected at ∼2 months before (1) and 2 months after (2) inoculation and at autopsy (3), and 1 μg of DNA from autopsy PBMCs, isolated splenocytes, and bone marrow obtained at 3.5 months after inoculation or from 50 μL of autopsy serum, were tested for WHV DNA by nested PCR using WHV C gene–specific primers and hybridization to WHV DNA probe. DNA from serum of a WHsAg-positive chronic carrier was included as a positive control, and water instead of DNA and a mock sample extracted in parallel with test samples were used negative controls. Positive samples showed the expected 428-bp nucleotide fragment noted by arrowhead.

Techniques Used: Expressing, Isolation, Nested PCR, Hybridization, Positive Control

Detection of WHV DNA in serial serum samples from 4B/M offspring. Sera collected between 2 and 36 months after birth were tested for WHV DNA by nested PCR using WHV C, S, and X gene–specific primers, and the amplified products were analyzed by Southern blot hybridization to recombinant WHV DNA probe. DNA isolated from serum of a WHsAg-positive chronic carrier and water were used as positive control and negative control, respectively. Positive samples show the expected sizes (bp) of the amplified nucleotide fragments noted by arrowheads.
Figure Legend Snippet: Detection of WHV DNA in serial serum samples from 4B/M offspring. Sera collected between 2 and 36 months after birth were tested for WHV DNA by nested PCR using WHV C, S, and X gene–specific primers, and the amplified products were analyzed by Southern blot hybridization to recombinant WHV DNA probe. DNA isolated from serum of a WHsAg-positive chronic carrier and water were used as positive control and negative control, respectively. Positive samples show the expected sizes (bp) of the amplified nucleotide fragments noted by arrowheads.

Techniques Used: Nested PCR, Amplification, Southern Blot, Hybridization, Recombinant, Isolation, Positive Control, Negative Control

3) Product Images from "Insights into the complex regulation of rpoS in Borrelia burgdorferi"

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

Journal: Molecular Microbiology

doi: 10.1111/j.1365-2958.2007.05813.x

Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.
Figure Legend Snippet: Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.

Techniques Used: Quantitative RT-PCR, Standard Deviation, Plasmid Preparation

Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).
Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

Techniques Used: Quantitative RT-PCR, Standard Deviation

Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.
Figure Legend Snippet: Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.

Techniques Used: Quantitative RT-PCR, Standard Deviation

Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.
Figure Legend Snippet: Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).
Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

Techniques Used: Quantitative RT-PCR, Standard Deviation

Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.
Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

Techniques Used: Quantitative RT-PCR, Standard Deviation

4) Product Images from "HIV Restriction by APOBEC3 in Humanized Mice"

Article Title: HIV Restriction by APOBEC3 in Humanized Mice

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003242

vif -deleted HIV-1 JR-CSF does not overcome APOBEC3 restriction in vivo . (A) Longitudinal analysis of plasma viral load in humanized mice intravenously infected with 9×10 4 TCIU of wild-type HIV-1 JR-CSF or 3.6×10 5 TCIU of HIV JR-CSF Δ vif . (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 17 viral DNA sequences amplified from the tissues of infected mice. Viral DNA from mice infected with HIV JR-CSF Δ vif had 25%–65% of G G sites mutated. (D) G to A mutational profile of all viral DNA from mice infected with HIV JR-CSF Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites. D4-D8, WT2-WT3, NSG-hu mice. D9-D11, NSG-BLT mice.
Figure Legend Snippet: vif -deleted HIV-1 JR-CSF does not overcome APOBEC3 restriction in vivo . (A) Longitudinal analysis of plasma viral load in humanized mice intravenously infected with 9×10 4 TCIU of wild-type HIV-1 JR-CSF or 3.6×10 5 TCIU of HIV JR-CSF Δ vif . (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 17 viral DNA sequences amplified from the tissues of infected mice. Viral DNA from mice infected with HIV JR-CSF Δ vif had 25%–65% of G G sites mutated. (D) G to A mutational profile of all viral DNA from mice infected with HIV JR-CSF Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites. D4-D8, WT2-WT3, NSG-hu mice. D9-D11, NSG-BLT mice.

Techniques Used: In Vivo, Mouse Assay, Infection, Nested PCR, Amplification, Mutagenesis

Restoration of vif occurs only following direct virus injection into the thymus. (A) Longitudinal analysis of plasma viral load in NSG-BLT humanized mice infected with HIV JR-CSF vif FS directly into the human thymic implant, spleen, liver, or lung. Solid symbols represent mice infected with a low dose of virus (9×10 4 TCIU), open symbols represent the high dose infection (3.6×10 5 TCIU). (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 44 viral DNA sequences amplified from the tissues of mice injected with HIV JR-CSF vif FS. Mice injected into the thymic implant had no G G to A G mutations in any tissue whereas HIV DNA from four tissues of mouse FS24 injected into the spleen is hypermutated. (D) G to A mutational profile of all viral DNA from mouse FS24 which failed to restore the vif ORF. Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites.
Figure Legend Snippet: Restoration of vif occurs only following direct virus injection into the thymus. (A) Longitudinal analysis of plasma viral load in NSG-BLT humanized mice infected with HIV JR-CSF vif FS directly into the human thymic implant, spleen, liver, or lung. Solid symbols represent mice infected with a low dose of virus (9×10 4 TCIU), open symbols represent the high dose infection (3.6×10 5 TCIU). (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 44 viral DNA sequences amplified from the tissues of mice injected with HIV JR-CSF vif FS. Mice injected into the thymic implant had no G G to A G mutations in any tissue whereas HIV DNA from four tissues of mouse FS24 injected into the spleen is hypermutated. (D) G to A mutational profile of all viral DNA from mouse FS24 which failed to restore the vif ORF. Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites.

Techniques Used: Injection, Mouse Assay, Infection, Nested PCR, Amplification, Mutagenesis

Human APOBEC3 exerts a strong selective pressure on HIV-1 JR-CSF containing a frameshift in vif . (A) Plasma viral load analysis in humanized mice intravenously infected with 9×10 4 or 3.6×10 5 TCIU of HIV JR-CSF vif FS. Viral RNA was not detected in the plasma (circles, n = 10) unless the vif ORF is restored (triangles, n = 6). The appearance of plasma viremia was delayed by 4 weeks in one of these mice. (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice intravenously infected with HIV JR-CSF vif FS. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Viral DNA is sparsely present in tissues from mice where vif was not restored (indicated as FS1–10). In contrast, all tissues analyzed from the six mice where vif was restored had viral DNA present (FS11–16). N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 76 viral DNA sequences amplified from the tissues of HIV JR-CSF vif FS infected mice where vif was not restored (40% of all G G sites mutated) or mice where vif was restored (no hypermutation). Data represent mean +/− SEM. (D) G to A mutational profile of all viral DNA from mice infected with HIV JR-CSF Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites. FS1–FS9, FS11–FS14, NSG-hu mice. FS10, FS15–FS16 NSG-BLT mice.
Figure Legend Snippet: Human APOBEC3 exerts a strong selective pressure on HIV-1 JR-CSF containing a frameshift in vif . (A) Plasma viral load analysis in humanized mice intravenously infected with 9×10 4 or 3.6×10 5 TCIU of HIV JR-CSF vif FS. Viral RNA was not detected in the plasma (circles, n = 10) unless the vif ORF is restored (triangles, n = 6). The appearance of plasma viremia was delayed by 4 weeks in one of these mice. (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice intravenously infected with HIV JR-CSF vif FS. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Viral DNA is sparsely present in tissues from mice where vif was not restored (indicated as FS1–10). In contrast, all tissues analyzed from the six mice where vif was restored had viral DNA present (FS11–16). N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 76 viral DNA sequences amplified from the tissues of HIV JR-CSF vif FS infected mice where vif was not restored (40% of all G G sites mutated) or mice where vif was restored (no hypermutation). Data represent mean +/− SEM. (D) G to A mutational profile of all viral DNA from mice infected with HIV JR-CSF Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites. FS1–FS9, FS11–FS14, NSG-hu mice. FS10, FS15–FS16 NSG-BLT mice.

Techniques Used: Mouse Assay, Infection, Nested PCR, Amplification, Mutagenesis

Human APOBEC3 rapidly restricts vif -deleted HIV-1 JR-CSF in vivo . (A) Replication of HIV JR-CSF , HIV JR-CSF Δ vif , and HIV JR-CSF vif FS in CEM-SS cells expressing CCR5 (CEM-SS CCR5). Culture supernatant was assayed for p24 Gag by ELISA at three day intervals to determine the replication kinetics of the mutant viruses. (B) Nested PCR amplification of viral DNA from the tissues obtained one week post-exposure from a representative NSG-hu mouse infected with 9×10 4 TCIU of wild-type HIV-1 JR-CSF (WT1) or from three mice infected with 3.6×10 5 TCIU of HIV JR-CSF Δ vif (indicated as D1–3). (C) Highlighter sequence analysis of 7 wild-type and 3 Δ vif HIV DNA sequences. Amplified viral DNA from panel A showed no APOBEC3 induced mutations in HIV JR-CSF (WT1 all sequence from tissues is shown together). In contrast, viral DNA from all positive tissues obtained from HIV JR-CSF Δ vif infected mice had G to A (green lines) and/or C to T mutations (red lines). HIV-1 JR-CSF nucleotide numbers are indicated at the bottom.
Figure Legend Snippet: Human APOBEC3 rapidly restricts vif -deleted HIV-1 JR-CSF in vivo . (A) Replication of HIV JR-CSF , HIV JR-CSF Δ vif , and HIV JR-CSF vif FS in CEM-SS cells expressing CCR5 (CEM-SS CCR5). Culture supernatant was assayed for p24 Gag by ELISA at three day intervals to determine the replication kinetics of the mutant viruses. (B) Nested PCR amplification of viral DNA from the tissues obtained one week post-exposure from a representative NSG-hu mouse infected with 9×10 4 TCIU of wild-type HIV-1 JR-CSF (WT1) or from three mice infected with 3.6×10 5 TCIU of HIV JR-CSF Δ vif (indicated as D1–3). (C) Highlighter sequence analysis of 7 wild-type and 3 Δ vif HIV DNA sequences. Amplified viral DNA from panel A showed no APOBEC3 induced mutations in HIV JR-CSF (WT1 all sequence from tissues is shown together). In contrast, viral DNA from all positive tissues obtained from HIV JR-CSF Δ vif infected mice had G to A (green lines) and/or C to T mutations (red lines). HIV-1 JR-CSF nucleotide numbers are indicated at the bottom.

Techniques Used: In Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Mutagenesis, Nested PCR, Amplification, Infection, Mouse Assay, Sequencing

Sustained Vif-independent replication of CXCR4 tropic HIV-1. (A) Plasma viral load was monitored in NSG-BLT humanized mice infected with 3.6×10 5 TCIU HIV LAI Δ vif directly into the human thymic implant, spleen, liver, or lung. Direct injection of HIV LAI Δ vif into the thymus resulted in plasma viremia in 4/4 infections. (B) Longitudinal analysis of plasma viral load in humanized mice infected intravenously with 3.6×10 5 TCIU of HIV LAI Δ vif (n = 7) or 3–9×10 4 TCIU of wild-type HIV LAI (n = 6). Data represent mean +/− SEM. (C) Longitudinal analysis of the percentage of CD4 + T cells in the peripheral blood of humanized mice infected in panel B. (D) Comparison of the G to A mutation frequency in the viral RNA from the plasma and the viral DNA from peripheral blood cells from mice intravenously infected with HIV LAI Δ vif . Data represent mean +/− SEM from 18 sequences, ** p = 0.0066. (E) Detection of HIV DNA (+) by nested PCR from the tissues of BLT humanized mice in panels A and B. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Direct injection of HIV LAI Δ vif into the liver, lung or spleen resulted in limited tissue distribution of viral DNA. (F) Comparison of the G to A mutation frequency in viral DNA from the thymus compared to viral DNA from other tissues of mice infected intrathymically (n = 4) and intravenously (n = 7) with HIV LAI Δ vif . Data represent mean +/− SEM from 83 sequences. (G) G to A mutational profile of all viral DNA from mice infected with HIV LAI Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites.
Figure Legend Snippet: Sustained Vif-independent replication of CXCR4 tropic HIV-1. (A) Plasma viral load was monitored in NSG-BLT humanized mice infected with 3.6×10 5 TCIU HIV LAI Δ vif directly into the human thymic implant, spleen, liver, or lung. Direct injection of HIV LAI Δ vif into the thymus resulted in plasma viremia in 4/4 infections. (B) Longitudinal analysis of plasma viral load in humanized mice infected intravenously with 3.6×10 5 TCIU of HIV LAI Δ vif (n = 7) or 3–9×10 4 TCIU of wild-type HIV LAI (n = 6). Data represent mean +/− SEM. (C) Longitudinal analysis of the percentage of CD4 + T cells in the peripheral blood of humanized mice infected in panel B. (D) Comparison of the G to A mutation frequency in the viral RNA from the plasma and the viral DNA from peripheral blood cells from mice intravenously infected with HIV LAI Δ vif . Data represent mean +/− SEM from 18 sequences, ** p = 0.0066. (E) Detection of HIV DNA (+) by nested PCR from the tissues of BLT humanized mice in panels A and B. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Direct injection of HIV LAI Δ vif into the liver, lung or spleen resulted in limited tissue distribution of viral DNA. (F) Comparison of the G to A mutation frequency in viral DNA from the thymus compared to viral DNA from other tissues of mice infected intrathymically (n = 4) and intravenously (n = 7) with HIV LAI Δ vif . Data represent mean +/− SEM from 83 sequences. (G) G to A mutational profile of all viral DNA from mice infected with HIV LAI Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites.

Techniques Used: Mouse Assay, Infection, Injection, Mutagenesis, Nested PCR, Amplification

5) Product Images from "Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells"

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells

Journal: Genes & Development

doi: 10.1101/gad.187328.112

Impaired B lymphopoiesis and expression of regulatory genes in Ebf1 fl/fl RERT Cre bone marrow. ( A ) Immunoblot analysis to detect Ebf1 and Stat5a in lysates of splenic B cells from mice that were treated with tamoxifen 4-OHT (3 mg) on two consecutive days and sacrificed on various days after first administration. ( B ) Flow cytometric analysis of bone marrow to detect B220 + CD43 + pro-B cells, B220 intermediate CD43 − pre-B cells, and B220 high CD43 − recirculating B cells. Numbers in quadrants indicate percentage of cells. ( C ) Flow cytometric analysis to detect differentiation and generation of CD25 + κ − pre-B cells and CD25 − κ + immature B cells from CD19 + CD43 + c-kit − pro-B cells in fetal liver cell cultures. Cells cultured in the presence of OP9 feeders and IL-7 were treated with 2 μM 4-OHT for 24 h and cultured for an additional 4 d. Differentiation was induced by withdrawal of IL-7. ( D ) Semiquantitative RT–PCR analysis of κ light chain GLTs in pro-B cells that were cultured in the presence of OP9 feeders and IL-7 for 6 d and treated with 2 μM 4-OHT during the first day of culture. Data are representative of at least four ( A ) or three ( B – D ) experiments. ( E ) Quantitative RT–PCR analysis to examine the expression of B-cell-specific regulatory genes in pro-B-cell cultures. Data represent mean values of three independent biological replicates, and the raw cycle values were normalized to actin expression. Error bars indicate standard deviation (SD). ( F ) Quantification of Ebf1 binding to target genes in 38B9 cells by ChIP and quantitative PCR analysis. Binding is represented as percentage of input chromatin, and error bars represent SD of duplicate ChIP experiments (see also Supplemental Fig. S1; Supplemental Table S1). ( G ) Sequence tag profiles in pro-B cells and splenic B cells at the Irf4 and Irf8 ) and splenic B cells (this study), as well as DNase I hypersensitivity sites (DHS) in CD19 + ) and splenic B cells (this study).
Figure Legend Snippet: Impaired B lymphopoiesis and expression of regulatory genes in Ebf1 fl/fl RERT Cre bone marrow. ( A ) Immunoblot analysis to detect Ebf1 and Stat5a in lysates of splenic B cells from mice that were treated with tamoxifen 4-OHT (3 mg) on two consecutive days and sacrificed on various days after first administration. ( B ) Flow cytometric analysis of bone marrow to detect B220 + CD43 + pro-B cells, B220 intermediate CD43 − pre-B cells, and B220 high CD43 − recirculating B cells. Numbers in quadrants indicate percentage of cells. ( C ) Flow cytometric analysis to detect differentiation and generation of CD25 + κ − pre-B cells and CD25 − κ + immature B cells from CD19 + CD43 + c-kit − pro-B cells in fetal liver cell cultures. Cells cultured in the presence of OP9 feeders and IL-7 were treated with 2 μM 4-OHT for 24 h and cultured for an additional 4 d. Differentiation was induced by withdrawal of IL-7. ( D ) Semiquantitative RT–PCR analysis of κ light chain GLTs in pro-B cells that were cultured in the presence of OP9 feeders and IL-7 for 6 d and treated with 2 μM 4-OHT during the first day of culture. Data are representative of at least four ( A ) or three ( B – D ) experiments. ( E ) Quantitative RT–PCR analysis to examine the expression of B-cell-specific regulatory genes in pro-B-cell cultures. Data represent mean values of three independent biological replicates, and the raw cycle values were normalized to actin expression. Error bars indicate standard deviation (SD). ( F ) Quantification of Ebf1 binding to target genes in 38B9 cells by ChIP and quantitative PCR analysis. Binding is represented as percentage of input chromatin, and error bars represent SD of duplicate ChIP experiments (see also Supplemental Fig. S1; Supplemental Table S1). ( G ) Sequence tag profiles in pro-B cells and splenic B cells at the Irf4 and Irf8 ) and splenic B cells (this study), as well as DNase I hypersensitivity sites (DHS) in CD19 + ) and splenic B cells (this study).

Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing

Roles of Ebf1 in peripheral B-cell subsets and BAFF-R-mediated cell survival. ( A ) Flow cytometric analysis of Ebf1 +/fl RERT Cre and Ebf1 fl/fl RERT Cre splenocytes to detect IgM hi IgD + immature B cells and IgM hi IgD hi mature B cells ( top panels), IgM hi IgD hi B220 + AA4.1 + transitional B cells ( middle panels), and B220 + CD23 − Cd21 hi MZ B cells and B220 + CD23 + CD21 + FO B cells ( bottom panels). Numbers in quadrants indicate percentage of cells. ( B ) Flow cytometric analysis of Ebf1 +/fl Cd21 Cre and Ebf1 fl/fl Cd21 Cre splenocytes to detect MZ B cells and FO B cells. ( C ) Quantitative RT–PCR analysis of Ebf1-regulated targets in sorted MZ B cells. Fold expression values are relative to heterozygote control cells. ( D ) ChIP analysis to examine binding of Ebf1 to the Tnfrsf13c (Baff-R) locus in 38B9 pro-B cells or MACS-enriched splenic B cells. Binding is represented as percentage of input chromatin. ( E ) Flow cytometric analysis of proliferation of splenic B cells that were depleted of non-FO B cells with antibodies directed against CD43, CD4, CD8a, Gr1, AA4.1, and CD9. Cells sorted 10 d after the initial 4-OHT treatment were CFSE-labeled and stimulated with LPS or αIgM F(ab′) 2 . Proliferation was determined by CFSE dilution 3 d after stimulation. Numbers indicate percentages of cells in the area marked by the thin bar. ( F ) Analysis of apoptosis of LPS- or α-IgM-stimulated splenic B cells at the specified days (d) ( n = 3). ( G ) Flow cytometric analysis of surface expression of BAFF-R in resting and stimulated splenic B cells. Cells were activated with the indicated stimuli for 40 h. The graph is representative of three experiments. ( H ) Analysis of the survival of Ebf1 +/fl RERT Cre and Ebf1 fl/fl RERT Cre resting splenic B cells in the presence of optimal (20 ng/mL) and limiting (2 ng/mL) exogenous BAFF ( n = 3).
Figure Legend Snippet: Roles of Ebf1 in peripheral B-cell subsets and BAFF-R-mediated cell survival. ( A ) Flow cytometric analysis of Ebf1 +/fl RERT Cre and Ebf1 fl/fl RERT Cre splenocytes to detect IgM hi IgD + immature B cells and IgM hi IgD hi mature B cells ( top panels), IgM hi IgD hi B220 + AA4.1 + transitional B cells ( middle panels), and B220 + CD23 − Cd21 hi MZ B cells and B220 + CD23 + CD21 + FO B cells ( bottom panels). Numbers in quadrants indicate percentage of cells. ( B ) Flow cytometric analysis of Ebf1 +/fl Cd21 Cre and Ebf1 fl/fl Cd21 Cre splenocytes to detect MZ B cells and FO B cells. ( C ) Quantitative RT–PCR analysis of Ebf1-regulated targets in sorted MZ B cells. Fold expression values are relative to heterozygote control cells. ( D ) ChIP analysis to examine binding of Ebf1 to the Tnfrsf13c (Baff-R) locus in 38B9 pro-B cells or MACS-enriched splenic B cells. Binding is represented as percentage of input chromatin. ( E ) Flow cytometric analysis of proliferation of splenic B cells that were depleted of non-FO B cells with antibodies directed against CD43, CD4, CD8a, Gr1, AA4.1, and CD9. Cells sorted 10 d after the initial 4-OHT treatment were CFSE-labeled and stimulated with LPS or αIgM F(ab′) 2 . Proliferation was determined by CFSE dilution 3 d after stimulation. Numbers indicate percentages of cells in the area marked by the thin bar. ( F ) Analysis of apoptosis of LPS- or α-IgM-stimulated splenic B cells at the specified days (d) ( n = 3). ( G ) Flow cytometric analysis of surface expression of BAFF-R in resting and stimulated splenic B cells. Cells were activated with the indicated stimuli for 40 h. The graph is representative of three experiments. ( H ) Analysis of the survival of Ebf1 +/fl RERT Cre and Ebf1 fl/fl RERT Cre resting splenic B cells in the presence of optimal (20 ng/mL) and limiting (2 ng/mL) exogenous BAFF ( n = 3).

Techniques Used: Flow Cytometry, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, Binding Assay, Magnetic Cell Separation, Labeling

Ebf1 regulates proliferative expansion and survival of pro-B cells. ( A ) Analysis of apoptosis of pro-B cells cultured in the presence of feeders and IL-7 after induced deletion of Ebf1 . Numbers of viable (annexin V- and PI-negative) cells were determined by flow cytometry at the indicated time points after 4-OHT treatment, and the data were converted to the percentage of viable cells at day 3. Line graphs represent the average survival of three independent cell cultures at various days (d). ( B , C ) Analysis of survival of primary Ebf1 fl/fl RERT Cre TgBcl2 pro-B cells ( n = 3) and transformed Ebf1 fl/fl RERT Cre ::A-MuLV pro-B cells ( n = 4), as described above. ( D , E ) Analysis of apoptosis in A-MuLV-transformed Ebf1 fl/fl pro-B cells that have been transduced with a GFP-expressing retrovirus or bicistronic retroviruses expressing both GFP and various Ebf1 target genes. Cells sorted for GFP expression were treated with 4-OHT, and the percentages of viable cells were determined at the indicated time points after treatment ( n = 4). Full and partial rescue of the survival defect of Ebf1-deficient cells is observed by expression of c-Myb and Bcl2l1, respectively. ( F ) Flow cytometric analysis of the proliferation of Ebf1 +/fl RERT Cre (thick line) and Ebf1 fl/fl RERT Cre (gray fill) pro-B cells treated with 2 μM 4-OHT for 24 h, cultured for 2 d without 4-OHT, labeled with CFSE, and assessed by CFSE dilution after 3 d ( n = 4). Numbers indicate the percentage of cells in the area marked by the thin bar. ( G , H ) Flow cytometric analysis of cell cycle progression of Ebf1 +/fl RERT Cre (black fill) and Ebf1 fl/fl RERT Cre (gray fill) primary pro-B ( G ) or A-MuLV-transformed pro-B ( H ) cell cultures 5 d after 4-OHT administration. Cells were fixed, stained with PI to assess DNA content, and gated for intact cells. Representative plot and quantitative analysis of cell cycle distribution are indicated ( n = 3). Numbers indicate percentage of cells. ( I ) Quantitative RT–PCR analysis to determine the expression of regulatory genes involved in cell cycle or survival and identified as indirect or direct (*) Ebf1 targets. Primary pro-B cells ( top panel) and A-MuLV-transformed pro-B cells ( bottom panel) were harvested 5 d after 4-OHT treatment. Data represent mean values of three biological replicates, and raw cycle values were normalized to actin . Ebf1 +/+ samples were set to 100%. ( J , K ) Immunoblot analysis to detect Cdc2 phosphorylation on Tyr 15 in primary ( J ) and A-MuLV-transformed ( K ) Ebf1 fl/fl RERT Cre pro-B cells. ( L ) Immunoblot analysis to detect Rb phosphorylation and expression of Ebf1, c-Myb, Cdc6, and Cdt1 in A-MuLV-transformed Ebf1 fl/fl RERT Cre pro-B cells.
Figure Legend Snippet: Ebf1 regulates proliferative expansion and survival of pro-B cells. ( A ) Analysis of apoptosis of pro-B cells cultured in the presence of feeders and IL-7 after induced deletion of Ebf1 . Numbers of viable (annexin V- and PI-negative) cells were determined by flow cytometry at the indicated time points after 4-OHT treatment, and the data were converted to the percentage of viable cells at day 3. Line graphs represent the average survival of three independent cell cultures at various days (d). ( B , C ) Analysis of survival of primary Ebf1 fl/fl RERT Cre TgBcl2 pro-B cells ( n = 3) and transformed Ebf1 fl/fl RERT Cre ::A-MuLV pro-B cells ( n = 4), as described above. ( D , E ) Analysis of apoptosis in A-MuLV-transformed Ebf1 fl/fl pro-B cells that have been transduced with a GFP-expressing retrovirus or bicistronic retroviruses expressing both GFP and various Ebf1 target genes. Cells sorted for GFP expression were treated with 4-OHT, and the percentages of viable cells were determined at the indicated time points after treatment ( n = 4). Full and partial rescue of the survival defect of Ebf1-deficient cells is observed by expression of c-Myb and Bcl2l1, respectively. ( F ) Flow cytometric analysis of the proliferation of Ebf1 +/fl RERT Cre (thick line) and Ebf1 fl/fl RERT Cre (gray fill) pro-B cells treated with 2 μM 4-OHT for 24 h, cultured for 2 d without 4-OHT, labeled with CFSE, and assessed by CFSE dilution after 3 d ( n = 4). Numbers indicate the percentage of cells in the area marked by the thin bar. ( G , H ) Flow cytometric analysis of cell cycle progression of Ebf1 +/fl RERT Cre (black fill) and Ebf1 fl/fl RERT Cre (gray fill) primary pro-B ( G ) or A-MuLV-transformed pro-B ( H ) cell cultures 5 d after 4-OHT administration. Cells were fixed, stained with PI to assess DNA content, and gated for intact cells. Representative plot and quantitative analysis of cell cycle distribution are indicated ( n = 3). Numbers indicate percentage of cells. ( I ) Quantitative RT–PCR analysis to determine the expression of regulatory genes involved in cell cycle or survival and identified as indirect or direct (*) Ebf1 targets. Primary pro-B cells ( top panel) and A-MuLV-transformed pro-B cells ( bottom panel) were harvested 5 d after 4-OHT treatment. Data represent mean values of three biological replicates, and raw cycle values were normalized to actin . Ebf1 +/+ samples were set to 100%. ( J , K ) Immunoblot analysis to detect Cdc2 phosphorylation on Tyr 15 in primary ( J ) and A-MuLV-transformed ( K ) Ebf1 fl/fl RERT Cre pro-B cells. ( L ) Immunoblot analysis to detect Rb phosphorylation and expression of Ebf1, c-Myb, Cdc6, and Cdt1 in A-MuLV-transformed Ebf1 fl/fl RERT Cre pro-B cells.

Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Transformation Assay, Transduction, Expressing, Labeling, Staining, Quantitative RT-PCR

Ebf1 regulates γ3 and γ1 germline transcription by binding to the IgH 3′ Eα LCR. ( A ) Schematic representation of the IgH locus. Light-gray bars depict rearranged variable gene segments, dark gray bars represent enhancers, and gray circles represent the promoters (P) of the constant region (C) gene segments. The bottom part depicts a magnified view of the γ3 gene segment and indicates regulatory sequences, switch (S) region, and 3′ Eα LCR. DHSs are marked with arrows and numbers. Numbers in squares represent HSs that are directly bound by Ebf1. ( B ) Semiquantitative RT–PCR analysis of IgH constant region GLTs in unstimulated B cells and B cells stimulated with LPS + IL4 or LPS + TGFβ for 16 h. ( C ) ChIP analysis to examine binding of Ebf1 in 38B9 pro-B cells or MACS-enriched splenic B cells. Binding is represented as percentage of input chromatin. (See also Supplemental Fig. S7.)
Figure Legend Snippet: Ebf1 regulates γ3 and γ1 germline transcription by binding to the IgH 3′ Eα LCR. ( A ) Schematic representation of the IgH locus. Light-gray bars depict rearranged variable gene segments, dark gray bars represent enhancers, and gray circles represent the promoters (P) of the constant region (C) gene segments. The bottom part depicts a magnified view of the γ3 gene segment and indicates regulatory sequences, switch (S) region, and 3′ Eα LCR. DHSs are marked with arrows and numbers. Numbers in squares represent HSs that are directly bound by Ebf1. ( B ) Semiquantitative RT–PCR analysis of IgH constant region GLTs in unstimulated B cells and B cells stimulated with LPS + IL4 or LPS + TGFβ for 16 h. ( C ) ChIP analysis to examine binding of Ebf1 in 38B9 pro-B cells or MACS-enriched splenic B cells. Binding is represented as percentage of input chromatin. (See also Supplemental Fig. S7.)

Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Magnetic Cell Separation

Impaired GC development in the spleens of Ebf1 fl/fl RERT Cre mice. ( A ) Flow cytometric analysis to detect GC B cells in the spleens of SRBC-immunized Ebf1 fl/fl RERT Cre and Ebf1 +/fl RERT Cre mice. Mice were immunized 8 d after the first 4-OHT treatment and were analyzed 6 d after immunization. Samples were gated on living B220 + cells, and the numbers on the FACS plots represent the percentage of PNA + FAS hi GC B cells ( n = 6). ( B ) Analysis of immune responses of Ebf1 fl/fl RERT Cre mice. Spleen sections were stained with PNA ( top panel) to reveal GC cells, and with antibodies to IgM (green) to reveal B-cell follicles and total IgG (green, bottom panel). Nuclei were stained with DAPI. ( C ) Gene expression analysis of sorted PNA + FAS hi GC B cells of the indicated genotypes by quantitative RT–PCR. Black and gray bars represent the averages of individually sorted samples ( n = 3). ( D ) Normal somatic hypermutation of Ebf1 fl/fl RERT Cre GC B cells upon immunization with SRBCs. Mice were immunized as described in A ; genomic DNA was extracted from sorted GC B cells and subjected to nested PCR amplification with two degenerate V and J segment primer pairs, and the amplified products corresponding to J H 3 were separated and cloned. The number of clones sequenced is shown in the circles in the middle of the pie diagrams; each diagram is representative of two independent experiments, with at least 40 cloned sequences each. ( E ) Relative titers of immunoglobulin subtypes in sera of Ebf1 +/fl RERT Cre mice (black triangles and blue lines represent individual samples and averages, respectively) and Ebf1 fl/fl RERT Cre mice (open circles and red lines represent individual samples and averages, respectively) at the specified days (d), determined by ELISA.
Figure Legend Snippet: Impaired GC development in the spleens of Ebf1 fl/fl RERT Cre mice. ( A ) Flow cytometric analysis to detect GC B cells in the spleens of SRBC-immunized Ebf1 fl/fl RERT Cre and Ebf1 +/fl RERT Cre mice. Mice were immunized 8 d after the first 4-OHT treatment and were analyzed 6 d after immunization. Samples were gated on living B220 + cells, and the numbers on the FACS plots represent the percentage of PNA + FAS hi GC B cells ( n = 6). ( B ) Analysis of immune responses of Ebf1 fl/fl RERT Cre mice. Spleen sections were stained with PNA ( top panel) to reveal GC cells, and with antibodies to IgM (green) to reveal B-cell follicles and total IgG (green, bottom panel). Nuclei were stained with DAPI. ( C ) Gene expression analysis of sorted PNA + FAS hi GC B cells of the indicated genotypes by quantitative RT–PCR. Black and gray bars represent the averages of individually sorted samples ( n = 3). ( D ) Normal somatic hypermutation of Ebf1 fl/fl RERT Cre GC B cells upon immunization with SRBCs. Mice were immunized as described in A ; genomic DNA was extracted from sorted GC B cells and subjected to nested PCR amplification with two degenerate V and J segment primer pairs, and the amplified products corresponding to J H 3 were separated and cloned. The number of clones sequenced is shown in the circles in the middle of the pie diagrams; each diagram is representative of two independent experiments, with at least 40 cloned sequences each. ( E ) Relative titers of immunoglobulin subtypes in sera of Ebf1 +/fl RERT Cre mice (black triangles and blue lines represent individual samples and averages, respectively) and Ebf1 fl/fl RERT Cre mice (open circles and red lines represent individual samples and averages, respectively) at the specified days (d), determined by ELISA.

Techniques Used: Mouse Assay, Flow Cytometry, FACS, Staining, Expressing, Quantitative RT-PCR, Nested PCR, Amplification, Clone Assay, Enzyme-linked Immunosorbent Assay

6) Product Images from "Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells"

Article Title: Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004797

Transcriptional mechanism of cadherin-11 regulation. A: MDA-MB-231 cells were grown at a medium density for 16 hours in the absence of serum. The cells were then pretreated with 5 μg/ml actinomycin D or an equivalent volume of ethanol (untreated). 30 minutes later the cells were treated with either 20 mM NaCl (control) or LiCl. At the indicated time points, RNA and protein were collect for real-time PCR analysis. B: MDA-MB-231 cells were treated with 20 mM NaCl or LiCl for 24 hours. Genomic DNA was then harvested for RNA polymerase II ChIP analysis followed by PCR specific to GAPDH and cadherin-11.
Figure Legend Snippet: Transcriptional mechanism of cadherin-11 regulation. A: MDA-MB-231 cells were grown at a medium density for 16 hours in the absence of serum. The cells were then pretreated with 5 μg/ml actinomycin D or an equivalent volume of ethanol (untreated). 30 minutes later the cells were treated with either 20 mM NaCl (control) or LiCl. At the indicated time points, RNA and protein were collect for real-time PCR analysis. B: MDA-MB-231 cells were treated with 20 mM NaCl or LiCl for 24 hours. Genomic DNA was then harvested for RNA polymerase II ChIP analysis followed by PCR specific to GAPDH and cadherin-11.

Techniques Used: Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction

β-catenin as a regulator of cadherin-11 expression. A: PC-3 cells were transfected with pGL3-CDH11-3′UTR NCBI or pGL3-CDH11-3′UTR Ensembl and pCMV-Renilla and along with either non-specific siRNA or siRNA directed against CTNNB1. 48 hours after transfection cells were were lysed using passive lysis buffer and luciferase activity was analyzed. Luciferase activity was normalized to renilla activity. (** indicates a p-value > 0.001). B: MDA-MB-231 and PC-3 cells were transfected with either non-specific scrambled siRNA (siScramble) or siRNA directed against CTNNB1 (si β-cat). 24 hours after transfection cells were treated with 1 μM BIO or meBIO (control). 48 hours after transfection protein was isolated for Western blot analysis. C: MDA-MB-231 and PC-3 cells were transfected with either non-specific scrambled siRNA (siScramble) or siRNA directed against CTNNB1 (si β-cat). 24 hours after transfection cells were treated with 1 μM BIO or meBIO (control). 48 hours after transfection RNA was isolated for real-time PCR analysis. (* indicates a p-value > 0.05; ** indicates a p-value > 0.005)
Figure Legend Snippet: β-catenin as a regulator of cadherin-11 expression. A: PC-3 cells were transfected with pGL3-CDH11-3′UTR NCBI or pGL3-CDH11-3′UTR Ensembl and pCMV-Renilla and along with either non-specific siRNA or siRNA directed against CTNNB1. 48 hours after transfection cells were were lysed using passive lysis buffer and luciferase activity was analyzed. Luciferase activity was normalized to renilla activity. (** indicates a p-value > 0.001). B: MDA-MB-231 and PC-3 cells were transfected with either non-specific scrambled siRNA (siScramble) or siRNA directed against CTNNB1 (si β-cat). 24 hours after transfection cells were treated with 1 μM BIO or meBIO (control). 48 hours after transfection protein was isolated for Western blot analysis. C: MDA-MB-231 and PC-3 cells were transfected with either non-specific scrambled siRNA (siScramble) or siRNA directed against CTNNB1 (si β-cat). 24 hours after transfection cells were treated with 1 μM BIO or meBIO (control). 48 hours after transfection RNA was isolated for real-time PCR analysis. (* indicates a p-value > 0.05; ** indicates a p-value > 0.005)

Techniques Used: Expressing, Transfection, Lysis, Luciferase, Activity Assay, Multiple Displacement Amplification, Isolation, Western Blot, Real-time Polymerase Chain Reaction

The stabilizing and destabilizing effects of the cadherin-11 3′UTR. A: PC-3, MDA-MB-231, and HEK 293 cells were transfected with pGL3-Promoter, pGL3-CDH11-3′UTR NCBI, or pGL3-CDH11-3′UTR Ensembl and pCMV-Renilla. 48 hours after transfection, cells were lysed using passive lysis buffer and luciferase activity was analyzed. (* indicates a p-value > 0.01; ** indicates a p-value > 0.001) B: MDA-MB-231 and PC-3 cells were transfected with pGL3-CDH11 3′-UTR and pCMV-Renilla along with either non-specfic siRNA (siScramble) or siRNA directed against Dicer (siDicer). 48 hours after transfection, cells were lysed using passive lysis buffer and luciferase activity was analyzed. (* indicates a p-value > 0.05) C and D: MDA-MB-231 cells were transfected with non-specific siRNA (siScramble), or siRNA directed against Dicer (si Dicer). Cells were collected for real-time PCR (C) and Western blot (D) analysis 72 hours after transfection.
Figure Legend Snippet: The stabilizing and destabilizing effects of the cadherin-11 3′UTR. A: PC-3, MDA-MB-231, and HEK 293 cells were transfected with pGL3-Promoter, pGL3-CDH11-3′UTR NCBI, or pGL3-CDH11-3′UTR Ensembl and pCMV-Renilla. 48 hours after transfection, cells were lysed using passive lysis buffer and luciferase activity was analyzed. (* indicates a p-value > 0.01; ** indicates a p-value > 0.001) B: MDA-MB-231 and PC-3 cells were transfected with pGL3-CDH11 3′-UTR and pCMV-Renilla along with either non-specfic siRNA (siScramble) or siRNA directed against Dicer (siDicer). 48 hours after transfection, cells were lysed using passive lysis buffer and luciferase activity was analyzed. (* indicates a p-value > 0.05) C and D: MDA-MB-231 cells were transfected with non-specific siRNA (siScramble), or siRNA directed against Dicer (si Dicer). Cells were collected for real-time PCR (C) and Western blot (D) analysis 72 hours after transfection.

Techniques Used: Multiple Displacement Amplification, Transfection, Lysis, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

In silico evaluation of the cadherin-11 3′UTR. A: Sequence of cadherin-11 3′-UTR according to the Ensembl database (NM_001797). Bolded sequences indicate the poly-A signals and site respectively. Blue highlighted sequences indicate Shaw-Kamens, destabilizing sequences. The first two red highlighted sequences indicate the primers used to design pGL3-CDH11-3′UTR NCBI, as denoted by the bracket. The first and last red highlighted sequences indicate the primers used to design pGL3-CDH11-3′UTR Ensembl, as denoted by the bracket. B: Predicted secondary structure of the Ensembl cadherin-11 3′UTR (as predicted by GeneBee). C: Predicted secondary structure of the Ensembl E-cadher 3′UTR (as predicted by GeneBee). D: RT-PCR of PC3 RNA using primers designed approximately every 500 bp.
Figure Legend Snippet: In silico evaluation of the cadherin-11 3′UTR. A: Sequence of cadherin-11 3′-UTR according to the Ensembl database (NM_001797). Bolded sequences indicate the poly-A signals and site respectively. Blue highlighted sequences indicate Shaw-Kamens, destabilizing sequences. The first two red highlighted sequences indicate the primers used to design pGL3-CDH11-3′UTR NCBI, as denoted by the bracket. The first and last red highlighted sequences indicate the primers used to design pGL3-CDH11-3′UTR Ensembl, as denoted by the bracket. B: Predicted secondary structure of the Ensembl cadherin-11 3′UTR (as predicted by GeneBee). C: Predicted secondary structure of the Ensembl E-cadher 3′UTR (as predicted by GeneBee). D: RT-PCR of PC3 RNA using primers designed approximately every 500 bp.

Techniques Used: In Silico, Sequencing, Reverse Transcription Polymerase Chain Reaction

Density increases cadherin-11 and GSK3β inhibitors decrease cadherin-11 expression. A and B: MDA-MB-231 cells were plated at two densities, 50% and 90% confluency, called low and high, respectively. The cells were allowed to grow overnight in serum-free medium. 24 hours after plating cells were treated with 20 mM NaCl (N) or 20 mM LiCl (L). 24 hours after treatment, RNA and protein were collected for real-time PCR (A) and Western (B) analysis. C: Cancer cells BT549, Hs578T, PC-3, and Kato-III were plated at a medium density and allowed to adhere. Cells were then treated with 20 mM LiCl or NaCl control. 48 hours after transfection protein was collected for Western blot analysis. D: MDA-MB-231 cells were plated at a medium density, serum starved overnight, and treated with 20 mM NaCl or 20 mM LiCl. 24 hours after treatment, the cells were washed once with PBS and maintained in serum-free medium containing only 20 mM NaCl. RNA was collected at the times indicated for real-time PCR analysis. E and F: MDA-MB-231 cells were plated at a medium density and serum starved overnight. 16 hours after plating, the cells were treated with 1 μM meBIO (control), 1 μM BIO, or 20 mM LiCl. RNA (E) or protein (F) was collected at the designated times and analyzed using real-time PCR (E) and Western blot analysis (F). G: MDA-MB-231 and PC-3 cells were transfected with either non-specific scrambled siRNA (siScramble), or siRNA directed against GSK3β (si GSK3). 48 hours after transfection protein was isolated for Western blot analysis.
Figure Legend Snippet: Density increases cadherin-11 and GSK3β inhibitors decrease cadherin-11 expression. A and B: MDA-MB-231 cells were plated at two densities, 50% and 90% confluency, called low and high, respectively. The cells were allowed to grow overnight in serum-free medium. 24 hours after plating cells were treated with 20 mM NaCl (N) or 20 mM LiCl (L). 24 hours after treatment, RNA and protein were collected for real-time PCR (A) and Western (B) analysis. C: Cancer cells BT549, Hs578T, PC-3, and Kato-III were plated at a medium density and allowed to adhere. Cells were then treated with 20 mM LiCl or NaCl control. 48 hours after transfection protein was collected for Western blot analysis. D: MDA-MB-231 cells were plated at a medium density, serum starved overnight, and treated with 20 mM NaCl or 20 mM LiCl. 24 hours after treatment, the cells were washed once with PBS and maintained in serum-free medium containing only 20 mM NaCl. RNA was collected at the times indicated for real-time PCR analysis. E and F: MDA-MB-231 cells were plated at a medium density and serum starved overnight. 16 hours after plating, the cells were treated with 1 μM meBIO (control), 1 μM BIO, or 20 mM LiCl. RNA (E) or protein (F) was collected at the designated times and analyzed using real-time PCR (E) and Western blot analysis (F). G: MDA-MB-231 and PC-3 cells were transfected with either non-specific scrambled siRNA (siScramble), or siRNA directed against GSK3β (si GSK3). 48 hours after transfection protein was isolated for Western blot analysis.

Techniques Used: Expressing, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Isolation

7) Product Images from "Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat"

Article Title: Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat

Journal: Plant Methods

doi: 10.1186/1746-4811-6-26

Silencing of BdPDS in B. distachyon leaves A Leaves of B. distachyon infected with BSMV-GFP 375 or BSMV-BdPDS. B BdPDS RNA expression levels in experiments 2 and 3 (Table 1) determined by qRT-PCR (normalization to 18 S rRNA). Plants were inoculated with either BSMV-BdPDS (black bars) or BSMV-GFP 375 (white bars). From left to right, the bars represent 16, 13, 6 and 6 samples, respectively. Error bars denote standard deviations. AU - Arbitrary units. Significantly differences: * ( p
Figure Legend Snippet: Silencing of BdPDS in B. distachyon leaves A Leaves of B. distachyon infected with BSMV-GFP 375 or BSMV-BdPDS. B BdPDS RNA expression levels in experiments 2 and 3 (Table 1) determined by qRT-PCR (normalization to 18 S rRNA). Plants were inoculated with either BSMV-BdPDS (black bars) or BSMV-GFP 375 (white bars). From left to right, the bars represent 16, 13, 6 and 6 samples, respectively. Error bars denote standard deviations. AU - Arbitrary units. Significantly differences: * ( p

Techniques Used: Infection, RNA Expression, Quantitative RT-PCR

Silencing of HvIPS1 in barley roots and shoots . A HvIPS1 RNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants inoculated with BSMV-IPS1 (black bars) or BSMV-GFP 250 (white bars). Plants were grown in 0 (5, 7, 9 dpi) or 1 mM Pi (9 dpi) and harvested at 5, 7, or 9 dpi. Each bar represents three samples (with one exception - only two samples for the 0 Pi, GFP, 9dpi bar). Error bars denote standard deviations. AU - Arbitrary units. B Stability of the BSMV-IPS1 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8, 9: 0 mM Pi, 9 dpi; 10, 11, 12: 1 mM Pi, 9 dpi; 13: plasmid containing BSMVγ-IPS1; 14: water control. M4: GeneRuler 50 bp DNA Ladder (Fermentas); white arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. C Stability of the BSMV-GFP 250 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8: 0 mM Pi, 9 dpi; 9, 10, 11: 1 mM Pi, 9 dpi. D HvIPS1 RNA expression levels in root and shoot tissue determined by qRT-PCR (normalization to ubiquitin). BSMV-IPS1 (black bars), BSMV-GFP 250 (white bars). Plant were grown in 0 mM Pi and harvested at 9 dpi. Each bar represents five samples. Error bars denote standard deviations.
Figure Legend Snippet: Silencing of HvIPS1 in barley roots and shoots . A HvIPS1 RNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants inoculated with BSMV-IPS1 (black bars) or BSMV-GFP 250 (white bars). Plants were grown in 0 (5, 7, 9 dpi) or 1 mM Pi (9 dpi) and harvested at 5, 7, or 9 dpi. Each bar represents three samples (with one exception - only two samples for the 0 Pi, GFP, 9dpi bar). Error bars denote standard deviations. AU - Arbitrary units. B Stability of the BSMV-IPS1 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8, 9: 0 mM Pi, 9 dpi; 10, 11, 12: 1 mM Pi, 9 dpi; 13: plasmid containing BSMVγ-IPS1; 14: water control. M4: GeneRuler 50 bp DNA Ladder (Fermentas); white arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. C Stability of the BSMV-GFP 250 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8: 0 mM Pi, 9 dpi; 9, 10, 11: 1 mM Pi, 9 dpi. D HvIPS1 RNA expression levels in root and shoot tissue determined by qRT-PCR (normalization to ubiquitin). BSMV-IPS1 (black bars), BSMV-GFP 250 (white bars). Plant were grown in 0 mM Pi and harvested at 9 dpi. Each bar represents five samples. Error bars denote standard deviations.

Techniques Used: RNA Expression, Quantitative RT-PCR, Construct, Polymerase Chain Reaction, Plasmid Preparation

BSMVγ constructs selected for relative stability also produce successful gene silencing in barley roots . A RT-PCR analysis of the stability of BSMVγ constructs in barley roots. Expected lengths for the PCR products are presented in brackets. Lanes 1, 2: BSMV-IPS1 (493 bp); 3, 4: BSMV-Pht1;1 (610 bp); 5, 6: BSMV-Pht1;4 (616 bp); 7, 8: BSMV-Pht1;7 (623 bp); 9, 10: BSMV-PHR1 (495 bp); 11, 12: BSMV-PHO2 247 (511 bp); 13, 14: BSMV-PHO2 387 (651 bp); 15, 16: GFP 250 (492 bp); 17: control plasmid carrying BSMVγ-GFP 375 (617 bp); 18: control plasmid carrying BSMVγ-IPS1 (493 bp); M4: GeneRuler 50 bp DNA Ladder (Fermentas); M5: O'GeneRuler 50bp DNA Ladder (Fermentas). White arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. B and C HvPHR1 expression levels in root tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHR1 (black bars), BSMV-GFP 250 (white bars). Samples in B are identical to those shown in A, lanes 9, 10 and 15,16. In C, each bar represents five independent samples. AU - arbitrary units; error bars denote standard deviations. D HvPHO2 expression levels in root and leaf tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHO2 247 (black bars), BSMV-GFP 250 (white bars). E As D, but bars represent Pi content in inoculated plants (in μmol Pi/g of fresh weight).
Figure Legend Snippet: BSMVγ constructs selected for relative stability also produce successful gene silencing in barley roots . A RT-PCR analysis of the stability of BSMVγ constructs in barley roots. Expected lengths for the PCR products are presented in brackets. Lanes 1, 2: BSMV-IPS1 (493 bp); 3, 4: BSMV-Pht1;1 (610 bp); 5, 6: BSMV-Pht1;4 (616 bp); 7, 8: BSMV-Pht1;7 (623 bp); 9, 10: BSMV-PHR1 (495 bp); 11, 12: BSMV-PHO2 247 (511 bp); 13, 14: BSMV-PHO2 387 (651 bp); 15, 16: GFP 250 (492 bp); 17: control plasmid carrying BSMVγ-GFP 375 (617 bp); 18: control plasmid carrying BSMVγ-IPS1 (493 bp); M4: GeneRuler 50 bp DNA Ladder (Fermentas); M5: O'GeneRuler 50bp DNA Ladder (Fermentas). White arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. B and C HvPHR1 expression levels in root tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHR1 (black bars), BSMV-GFP 250 (white bars). Samples in B are identical to those shown in A, lanes 9, 10 and 15,16. In C, each bar represents five independent samples. AU - arbitrary units; error bars denote standard deviations. D HvPHO2 expression levels in root and leaf tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHO2 247 (black bars), BSMV-GFP 250 (white bars). E As D, but bars represent Pi content in inoculated plants (in μmol Pi/g of fresh weight).

Techniques Used: Construct, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation, Expressing, Quantitative RT-PCR

Short inverted repeats are not inherently more stable or efficient for BSMV induced VIGS in barley roots . A HvCel1 RNA expression levels in root tissue determined by qRT-PCR (normalization to 18 S rRNA). Plants were inoculated with either BSMV-Cel1-1, BSMV-Cel1-3, BSMV-Cel1-IR, or BSMV-GFP 375 . Each bar represents five samples, each consisting of one plant in a pot. Error bars denote standard deviations. AU - Arbitrary units. B and C Stability of the BSMV constructs in roots of infected plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. B lanes 1-5: plants infected with BSMV-Cel1-1 (643 bp); lane 6: plasmid containing BSMV-Cel1-1 (643 bp); lane 7: plasmid containing BSMV without insert (242 bp); lanes 8-12: plants infected with BSMV-Cel1-3 (641 bp). C lanes 1-5: plants infected with BSMV-Cel1-IR (388 bp); lane 6: plasmid containing BSMV-Cel1-IR (388 bp); lane 7: plasmid containing BSMV without insert (242 bp); lane 8: plasmid containing BSMV-GFP 375 (617 bp); lanes 9-12: plants infected with BSMV-GFP 375 (617 bp). M5: O'Gene Ruler 50 bp DNA ladder (Fermentas). Expected lengths for the PCR products are given in brackets.
Figure Legend Snippet: Short inverted repeats are not inherently more stable or efficient for BSMV induced VIGS in barley roots . A HvCel1 RNA expression levels in root tissue determined by qRT-PCR (normalization to 18 S rRNA). Plants were inoculated with either BSMV-Cel1-1, BSMV-Cel1-3, BSMV-Cel1-IR, or BSMV-GFP 375 . Each bar represents five samples, each consisting of one plant in a pot. Error bars denote standard deviations. AU - Arbitrary units. B and C Stability of the BSMV constructs in roots of infected plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. B lanes 1-5: plants infected with BSMV-Cel1-1 (643 bp); lane 6: plasmid containing BSMV-Cel1-1 (643 bp); lane 7: plasmid containing BSMV without insert (242 bp); lanes 8-12: plants infected with BSMV-Cel1-3 (641 bp). C lanes 1-5: plants infected with BSMV-Cel1-IR (388 bp); lane 6: plasmid containing BSMV-Cel1-IR (388 bp); lane 7: plasmid containing BSMV without insert (242 bp); lane 8: plasmid containing BSMV-GFP 375 (617 bp); lanes 9-12: plants infected with BSMV-GFP 375 (617 bp). M5: O'Gene Ruler 50 bp DNA ladder (Fermentas). Expected lengths for the PCR products are given in brackets.

Techniques Used: RNA Expression, Quantitative RT-PCR, Construct, Infection, Polymerase Chain Reaction, Plasmid Preparation

Attempt at silencing HvPht1;1 expression in barley roots . A HvPht1;1 mRNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants were inoculated with either BSMV-Pht1;1 (black bars) or BSMV-GFP 375 (white bars). Plants were grown in hydroponic culture containing 0 or 1 mM Pi. Each bar represents three samples, error bars denote standard deviations. AU - Arbitrary units. B Schematic view of BSMVγ with introduced insert. Arrows show the position of the primers BSMVgbF and BSMVgbR used for assessing stability of insert. The length of virus sequence 5' and 3' of the insert is shown. PCR products of
Figure Legend Snippet: Attempt at silencing HvPht1;1 expression in barley roots . A HvPht1;1 mRNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants were inoculated with either BSMV-Pht1;1 (black bars) or BSMV-GFP 375 (white bars). Plants were grown in hydroponic culture containing 0 or 1 mM Pi. Each bar represents three samples, error bars denote standard deviations. AU - Arbitrary units. B Schematic view of BSMVγ with introduced insert. Arrows show the position of the primers BSMVgbF and BSMVgbR used for assessing stability of insert. The length of virus sequence 5' and 3' of the insert is shown. PCR products of "USER cloning" BSMV vectors are longer by 22 bp compared with restriction enzymes cloning. Not drawn to scale. C Stability of the BSMV-Pht1;1 and BSMV-GFP 375 constructs in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert as shown in B. Lanes 1, 2, 3: BSMV-Pht1;1, 0 mM Pi (610 bp); 4, 5, 6: BSMV-Pht1;1, 1 mM Pi (610 bp); 7, 8, 9: BSMV-GFP 375 , 0 mM Pi (617 bp); 10, 11, 12: BSMV-GFP 375 ,1 mM Pi (617 bp); 13: plasmid containing BSMVγ-PDS cassette (643 bp); 14: water control; M2: DNA marker; black arrow represents DNA fragment of 564 bp. The expected lengths for PCR products are given in brackets.

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Polymerase Chain Reaction, Clone Assay, Construct, Plasmid Preparation, Marker

8) Product Images from "Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis"

Article Title: Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis

Journal: Oncotarget

doi: 10.18632/oncotarget.16566

Transcriptional corepressors are recruited onto MLK3 promoter ( A ) Treatment regimens for ChIP assays. ( B ) The primary ChIP was performed with anti-ERα antibody, and re-immunoprecipitated second time with antibodies against NcoR or SMRT or LCoR. Final immunocomplexes were eluted in elution buffer and processed for PCR.
Figure Legend Snippet: Transcriptional corepressors are recruited onto MLK3 promoter ( A ) Treatment regimens for ChIP assays. ( B ) The primary ChIP was performed with anti-ERα antibody, and re-immunoprecipitated second time with antibodies against NcoR or SMRT or LCoR. Final immunocomplexes were eluted in elution buffer and processed for PCR.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction

MLK3 expression is regulated by E2-ER axis: the MLK3 mRNA expression levels were quantified by Real Time PCR ( A ) MLK3 mRNA levels in MCF7 (ER+) were compared to MDA-MB-231(ER − ) cells, using two pairs of specific primer sets. ( B ) MLK3 mRNA levels in isogenic T47D cell lines: A18; (ER + ) and C42; (ER − ) were determined using same set of primers, like in A. ( C ) The ER+ MCF7, T47D and ZR75-1 breast cancer cell lines were treated with E2 (10 nM) in phenol red free medium containing 0.2% charcoal stripped serum for different periods of time as indicated. Lysate were prepared in RIPA buffer and immunoblotted with anti-MLK3 antibody. GAPDH was used as a loading control.
Figure Legend Snippet: MLK3 expression is regulated by E2-ER axis: the MLK3 mRNA expression levels were quantified by Real Time PCR ( A ) MLK3 mRNA levels in MCF7 (ER+) were compared to MDA-MB-231(ER − ) cells, using two pairs of specific primer sets. ( B ) MLK3 mRNA levels in isogenic T47D cell lines: A18; (ER + ) and C42; (ER − ) were determined using same set of primers, like in A. ( C ) The ER+ MCF7, T47D and ZR75-1 breast cancer cell lines were treated with E2 (10 nM) in phenol red free medium containing 0.2% charcoal stripped serum for different periods of time as indicated. Lysate were prepared in RIPA buffer and immunoblotted with anti-MLK3 antibody. GAPDH was used as a loading control.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification

Estrogen Receptor regulates MLK3 promoter upon E2 treatment ( A ) schematic representation of MLK3 promoter cloned into pGL4.16 [luc2CP/Hygro] vector. Two putative EREs (hexagon) designated as proximal and distal are shown. Sequence of ERE binding sites with respective consensus sequences are shown below. A series of 5′ and 3′ progressive deletion mutants were generated by nested PCR using MLK3-P1 (−2993/+125) as a template. Five deletions mutants were designated: MLK3-P2 (−2993/−1293), MLK3-P3 (−1419/+125), MLK3-P4 (−2993/−1937), MLK3-P5 (−1960/−945) and MLK3-P6 (−296/+125). Position of EREs is indicated with reference to translation start codon ATG. Promoter activity was assayed in ( B ) ER + , MCF7 and ( C ) ER − , MDA-MB-231 cell lines as described in Materials and Methods. E2 (10 nM) or ethanol (vehicle) were added to the medium after 14 h of transfection. Cells were harvested 36 h after transfection. Luciferase activities were measured using the Dual Luciferase Assay Kit (Promega). Values are the mean ± S.D. for at least three independent experiments. *p
Figure Legend Snippet: Estrogen Receptor regulates MLK3 promoter upon E2 treatment ( A ) schematic representation of MLK3 promoter cloned into pGL4.16 [luc2CP/Hygro] vector. Two putative EREs (hexagon) designated as proximal and distal are shown. Sequence of ERE binding sites with respective consensus sequences are shown below. A series of 5′ and 3′ progressive deletion mutants were generated by nested PCR using MLK3-P1 (−2993/+125) as a template. Five deletions mutants were designated: MLK3-P2 (−2993/−1293), MLK3-P3 (−1419/+125), MLK3-P4 (−2993/−1937), MLK3-P5 (−1960/−945) and MLK3-P6 (−296/+125). Position of EREs is indicated with reference to translation start codon ATG. Promoter activity was assayed in ( B ) ER + , MCF7 and ( C ) ER − , MDA-MB-231 cell lines as described in Materials and Methods. E2 (10 nM) or ethanol (vehicle) were added to the medium after 14 h of transfection. Cells were harvested 36 h after transfection. Luciferase activities were measured using the Dual Luciferase Assay Kit (Promega). Values are the mean ± S.D. for at least three independent experiments. *p

Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Binding Assay, Generated, Nested PCR, Activity Assay, Multiple Displacement Amplification, Transfection, Luciferase

ERα and ERβ directly interact with EREs on MLK3 promoter ( A ) In vitro translated human ERα and ERβ receptors were incubated with P 32 -labeled proximal and distal EREs oligonucleotides and complexes were separated by electrophoresis. An oligo containing 3 copies of vitellogenin gene ERE (3X ERE) in tandem was used as a positive control. Free probes are shown at the bottom of gel. ( B ) Competitive EMSA was performed to confirm specific binding of ERα (lane 4–7) and ERβ (lane 8-11) to the distal ERE. The 25-, 50- or 100-fold molar excess of unlabeled oligonucleotide were added into tubes containing labeled distal ERE. ( C ) Chromatin extracts were prepared from MCF7 cells treated with E2 (10 nM) or ICI-182, 780 (20 nM) alone or together, as indicated. Formalin fixed chromatin was immunoprecipitated with specific antibodies. Amounts of co-precipitated DNA and the corresponding amount in the input chromatin samples were measured by PCR using primers that were designed in the region flanking EREs. Co-immunoprecipitation with IgG served as a negative control.
Figure Legend Snippet: ERα and ERβ directly interact with EREs on MLK3 promoter ( A ) In vitro translated human ERα and ERβ receptors were incubated with P 32 -labeled proximal and distal EREs oligonucleotides and complexes were separated by electrophoresis. An oligo containing 3 copies of vitellogenin gene ERE (3X ERE) in tandem was used as a positive control. Free probes are shown at the bottom of gel. ( B ) Competitive EMSA was performed to confirm specific binding of ERα (lane 4–7) and ERβ (lane 8-11) to the distal ERE. The 25-, 50- or 100-fold molar excess of unlabeled oligonucleotide were added into tubes containing labeled distal ERE. ( C ) Chromatin extracts were prepared from MCF7 cells treated with E2 (10 nM) or ICI-182, 780 (20 nM) alone or together, as indicated. Formalin fixed chromatin was immunoprecipitated with specific antibodies. Amounts of co-precipitated DNA and the corresponding amount in the input chromatin samples were measured by PCR using primers that were designed in the region flanking EREs. Co-immunoprecipitation with IgG served as a negative control.

Techniques Used: In Vitro, Incubation, Labeling, Electrophoresis, Positive Control, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Negative Control

9) Product Images from "Mice Lacking Hbp1 Function Are Viable and Fertile"

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile

Journal: PLoS ONE

doi: 10.1371/journal.pone.0170576

Immunohistochemical and gene expression analysis of Hbp1 +/+ and Hbp1 -/- embryonic gonads. (A) Proliferation marker Ki67 in germ cells (MVH-positive cells) was detected in both Hbp1 +/+ and Hbp1 -/- gonads at 12.5 dpc, but was absent in germ cells of 14.5 and 16.5 dpc gonads of both genotypes. Pluripotency marker OCT3/4 was expressed similarly in germ cells (MVH-positive cells) in Hbp1 +/+ and Hbp1 -/- gonads at 14.5 dpc and was undetectable at 16.5 dpc. Scale bar = 50μm. (B) qRT-PCR analysis of Hbp1 +/+ and Hbp1 -/- 16.5 dpc gonad samples revealed comparable expression between of various germ cell and somatic cell markers. Germ cell markers: Mvh , Oct3/4 and G 1 /G 0 arrest indicator p63 , somatic cell markers Fgf9 and Sox9 , Retinoblastoma family members Rb1 , p130 and p107 , and cell cycle regulators p21 , Ccnd1-3 and p53 displayed no significant difference between Hbp1 +/- and Hbp1 -/- samples. Samples normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate). * P
Figure Legend Snippet: Immunohistochemical and gene expression analysis of Hbp1 +/+ and Hbp1 -/- embryonic gonads. (A) Proliferation marker Ki67 in germ cells (MVH-positive cells) was detected in both Hbp1 +/+ and Hbp1 -/- gonads at 12.5 dpc, but was absent in germ cells of 14.5 and 16.5 dpc gonads of both genotypes. Pluripotency marker OCT3/4 was expressed similarly in germ cells (MVH-positive cells) in Hbp1 +/+ and Hbp1 -/- gonads at 14.5 dpc and was undetectable at 16.5 dpc. Scale bar = 50μm. (B) qRT-PCR analysis of Hbp1 +/+ and Hbp1 -/- 16.5 dpc gonad samples revealed comparable expression between of various germ cell and somatic cell markers. Germ cell markers: Mvh , Oct3/4 and G 1 /G 0 arrest indicator p63 , somatic cell markers Fgf9 and Sox9 , Retinoblastoma family members Rb1 , p130 and p107 , and cell cycle regulators p21 , Ccnd1-3 and p53 displayed no significant difference between Hbp1 +/- and Hbp1 -/- samples. Samples normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate). * P

Techniques Used: Immunohistochemistry, Expressing, Marker, Quantitative RT-PCR

Hbp1 expression in Rb +/+ and Rb -/- mutant XY gonads. Using qRT-PCR analysis, normalizing gene expression to 18S RNA, both Fl-Hbp1 (A) and ΔHbp1 (B) gene expression was significantly increased in Rb -/- mutant XY gonads at 14.5 dpc. In 16.5 dpc Rb -/- cultured XY gonads, there was no significant difference in either Fl-Hbp1 or ΔHbp1 gene expression, although germ cell marker Mvh was significantly increased at this timepoint ( C ). When gene expression was normalized to germ cell marker Mvh , both Fl-Hbp1 (D) and ΔHbp1 (E) gene expression was significantly decreased in 16.5 dpc Rb -/- cultured XY gonads. (mean ± S.E.M of three independent experiments, each performed in triplicate; wildtype controls ( Rb +/+ ) set to 1). * P
Figure Legend Snippet: Hbp1 expression in Rb +/+ and Rb -/- mutant XY gonads. Using qRT-PCR analysis, normalizing gene expression to 18S RNA, both Fl-Hbp1 (A) and ΔHbp1 (B) gene expression was significantly increased in Rb -/- mutant XY gonads at 14.5 dpc. In 16.5 dpc Rb -/- cultured XY gonads, there was no significant difference in either Fl-Hbp1 or ΔHbp1 gene expression, although germ cell marker Mvh was significantly increased at this timepoint ( C ). When gene expression was normalized to germ cell marker Mvh , both Fl-Hbp1 (D) and ΔHbp1 (E) gene expression was significantly decreased in 16.5 dpc Rb -/- cultured XY gonads. (mean ± S.E.M of three independent experiments, each performed in triplicate; wildtype controls ( Rb +/+ ) set to 1). * P

Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Cell Culture, Marker

Detection of Fl-Hbp1 and ΔHbp1 transcripts in wildtype and W e /W e gonads. (A) Detection of Fl-Hbp1 and ΔHbp1 in 14.5 dpc XX and XY gonads using whole mount in situ hybridisation (B) qRT-PCR analysis detected lower levels of Fl-Hbp1 and ΔHbp1 gene expression in 13.5 dpc XY and XX W e /W e mutant gonads which lack germ cells. Expression was normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate) and wildtype controls set to 1. * P
Figure Legend Snippet: Detection of Fl-Hbp1 and ΔHbp1 transcripts in wildtype and W e /W e gonads. (A) Detection of Fl-Hbp1 and ΔHbp1 in 14.5 dpc XX and XY gonads using whole mount in situ hybridisation (B) qRT-PCR analysis detected lower levels of Fl-Hbp1 and ΔHbp1 gene expression in 13.5 dpc XY and XX W e /W e mutant gonads which lack germ cells. Expression was normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate) and wildtype controls set to 1. * P

Techniques Used: In Situ, Hybridization, Quantitative RT-PCR, Expressing, Mutagenesis

Alternative splicing of Hbp1 transcripts. (A) Gene structure of the two Hbp1 splice variants. The full-length transcript comprises 11 exons and the truncated transcript is composed of the first 9 exons. Both transcripts contain identical 5’UTRs in addition to distinct 3’UTRs (open boxes), the specific riboprobes and real time PCR probes are depicted. (B) Location of HBP1 protein domains and nuclear localisation signals (NLS).
Figure Legend Snippet: Alternative splicing of Hbp1 transcripts. (A) Gene structure of the two Hbp1 splice variants. The full-length transcript comprises 11 exons and the truncated transcript is composed of the first 9 exons. Both transcripts contain identical 5’UTRs in addition to distinct 3’UTRs (open boxes), the specific riboprobes and real time PCR probes are depicted. (B) Location of HBP1 protein domains and nuclear localisation signals (NLS).

Techniques Used: Real-time Polymerase Chain Reaction

10) Product Images from "Mice Lacking Hbp1 Function Are Viable and Fertile"

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile

Journal: PLoS ONE

doi: 10.1371/journal.pone.0170576

Immunohistochemical and gene expression analysis of Hbp1 +/+ and Hbp1 -/- embryonic gonads. (A) Proliferation marker Ki67 in germ cells (MVH-positive cells) was detected in both Hbp1 +/+ and Hbp1 -/- gonads at 12.5 dpc, but was absent in germ cells of 14.5 and 16.5 dpc gonads of both genotypes. Pluripotency marker OCT3/4 was expressed similarly in germ cells (MVH-positive cells) in Hbp1 +/+ and Hbp1 -/- gonads at 14.5 dpc and was undetectable at 16.5 dpc. Scale bar = 50μm. (B) qRT-PCR analysis of Hbp1 +/+ and Hbp1 -/- 16.5 dpc gonad samples revealed comparable expression between of various germ cell and somatic cell markers. Germ cell markers: Mvh , Oct3/4 and G 1 /G 0 arrest indicator p63 , somatic cell markers Fgf9 and Sox9 , Retinoblastoma family members Rb1 , p130 and p107 , and cell cycle regulators p21 , Ccnd1-3 and p53 displayed no significant difference between Hbp1 +/- and Hbp1 -/- samples. Samples normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate). * P
Figure Legend Snippet: Immunohistochemical and gene expression analysis of Hbp1 +/+ and Hbp1 -/- embryonic gonads. (A) Proliferation marker Ki67 in germ cells (MVH-positive cells) was detected in both Hbp1 +/+ and Hbp1 -/- gonads at 12.5 dpc, but was absent in germ cells of 14.5 and 16.5 dpc gonads of both genotypes. Pluripotency marker OCT3/4 was expressed similarly in germ cells (MVH-positive cells) in Hbp1 +/+ and Hbp1 -/- gonads at 14.5 dpc and was undetectable at 16.5 dpc. Scale bar = 50μm. (B) qRT-PCR analysis of Hbp1 +/+ and Hbp1 -/- 16.5 dpc gonad samples revealed comparable expression between of various germ cell and somatic cell markers. Germ cell markers: Mvh , Oct3/4 and G 1 /G 0 arrest indicator p63 , somatic cell markers Fgf9 and Sox9 , Retinoblastoma family members Rb1 , p130 and p107 , and cell cycle regulators p21 , Ccnd1-3 and p53 displayed no significant difference between Hbp1 +/- and Hbp1 -/- samples. Samples normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate). * P

Techniques Used: Immunohistochemistry, Expressing, Marker, Quantitative RT-PCR

Hbp1 expression in Rb +/+ and Rb -/- mutant XY gonads. Using qRT-PCR analysis, normalizing gene expression to 18S RNA, both Fl-Hbp1 (A) and ΔHbp1 (B) gene expression was significantly increased in Rb -/- mutant XY gonads at 14.5 dpc. In 16.5 dpc Rb -/- cultured XY gonads, there was no significant difference in either Fl-Hbp1 or ΔHbp1 gene expression, although germ cell marker Mvh was significantly increased at this timepoint ( C ). When gene expression was normalized to germ cell marker Mvh , both Fl-Hbp1 (D) and ΔHbp1 (E) gene expression was significantly decreased in 16.5 dpc Rb -/- cultured XY gonads. (mean ± S.E.M of three independent experiments, each performed in triplicate; wildtype controls ( Rb +/+ ) set to 1). * P
Figure Legend Snippet: Hbp1 expression in Rb +/+ and Rb -/- mutant XY gonads. Using qRT-PCR analysis, normalizing gene expression to 18S RNA, both Fl-Hbp1 (A) and ΔHbp1 (B) gene expression was significantly increased in Rb -/- mutant XY gonads at 14.5 dpc. In 16.5 dpc Rb -/- cultured XY gonads, there was no significant difference in either Fl-Hbp1 or ΔHbp1 gene expression, although germ cell marker Mvh was significantly increased at this timepoint ( C ). When gene expression was normalized to germ cell marker Mvh , both Fl-Hbp1 (D) and ΔHbp1 (E) gene expression was significantly decreased in 16.5 dpc Rb -/- cultured XY gonads. (mean ± S.E.M of three independent experiments, each performed in triplicate; wildtype controls ( Rb +/+ ) set to 1). * P

Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Cell Culture, Marker

Detection of Fl-Hbp1 and ΔHbp1 transcripts in wildtype and W e /W e gonads. (A) Detection of Fl-Hbp1 and ΔHbp1 in 14.5 dpc XX and XY gonads using whole mount in situ hybridisation (B) qRT-PCR analysis detected lower levels of Fl-Hbp1 and ΔHbp1 gene expression in 13.5 dpc XY and XX W e /W e mutant gonads which lack germ cells. Expression was normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate) and wildtype controls set to 1. * P
Figure Legend Snippet: Detection of Fl-Hbp1 and ΔHbp1 transcripts in wildtype and W e /W e gonads. (A) Detection of Fl-Hbp1 and ΔHbp1 in 14.5 dpc XX and XY gonads using whole mount in situ hybridisation (B) qRT-PCR analysis detected lower levels of Fl-Hbp1 and ΔHbp1 gene expression in 13.5 dpc XY and XX W e /W e mutant gonads which lack germ cells. Expression was normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate) and wildtype controls set to 1. * P

Techniques Used: In Situ, Hybridization, Quantitative RT-PCR, Expressing, Mutagenesis

Alternative splicing of Hbp1 transcripts. (A) Gene structure of the two Hbp1 splice variants. The full-length transcript comprises 11 exons and the truncated transcript is composed of the first 9 exons. Both transcripts contain identical 5’UTRs in addition to distinct 3’UTRs (open boxes), the specific riboprobes and real time PCR probes are depicted. (B) Location of HBP1 protein domains and nuclear localisation signals (NLS).
Figure Legend Snippet: Alternative splicing of Hbp1 transcripts. (A) Gene structure of the two Hbp1 splice variants. The full-length transcript comprises 11 exons and the truncated transcript is composed of the first 9 exons. Both transcripts contain identical 5’UTRs in addition to distinct 3’UTRs (open boxes), the specific riboprobes and real time PCR probes are depicted. (B) Location of HBP1 protein domains and nuclear localisation signals (NLS).

Techniques Used: Real-time Polymerase Chain Reaction

11) Product Images from "Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues"

Article Title: Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200311057

Ecdysone-mediated dronc transcription is partially cycloheximide sensitive. (A) 10 7 l(2)mbn cells were treated with 10 μM ecdysone (Ecd) for the indicated time in the presence or absence (Control) of 10 μg/ml cycloheximide (CHX). RNA extracted from cells was analyzed by RT-PCR. (B) Northern blot analysis was performed on RNA samples from cells treated as in A. Where indicated, cells were treated with cycloheximide (CHX) for 2 h.
Figure Legend Snippet: Ecdysone-mediated dronc transcription is partially cycloheximide sensitive. (A) 10 7 l(2)mbn cells were treated with 10 μM ecdysone (Ecd) for the indicated time in the presence or absence (Control) of 10 μg/ml cycloheximide (CHX). RNA extracted from cells was analyzed by RT-PCR. (B) Northern blot analysis was performed on RNA samples from cells treated as in A. Where indicated, cells were treated with cycloheximide (CHX) for 2 h.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

l(2)mbn cells express the EcR-B1 isoform, which binds to the dronc promoter. (A) 10 7 l(2)mbn cells were treated with 10 μM ecdysone for the indicated time. RNA was extracted from cells and analyzed by RT-PCR to detect specific EcR isoforms. The last lane of the gel shows EcR isoforms expressed in late third instar larvae/prepupae (120 h after egg laying). (B) 9 μg of nuclear extracts prepared from l(2)mbn cells treated with ecdysone for 6 h was incubated with the dronc EcRBE or the EcRBE mutant probe for 20 min in the presence of 2 μl of EcR common, EcR-B1, or EcR-A antibody. A mouse control antibody was also used. Complexes were resolved on an acrylamide/TBE gel, dried on 3 MM Whatmann paper, and exposed to Kodak film overnight. EcR–Usp complex (EcR) and supershifted EcR–Usp complex (ss) are indicated.
Figure Legend Snippet: l(2)mbn cells express the EcR-B1 isoform, which binds to the dronc promoter. (A) 10 7 l(2)mbn cells were treated with 10 μM ecdysone for the indicated time. RNA was extracted from cells and analyzed by RT-PCR to detect specific EcR isoforms. The last lane of the gel shows EcR isoforms expressed in late third instar larvae/prepupae (120 h after egg laying). (B) 9 μg of nuclear extracts prepared from l(2)mbn cells treated with ecdysone for 6 h was incubated with the dronc EcRBE or the EcRBE mutant probe for 20 min in the presence of 2 μl of EcR common, EcR-B1, or EcR-A antibody. A mouse control antibody was also used. Complexes were resolved on an acrylamide/TBE gel, dried on 3 MM Whatmann paper, and exposed to Kodak film overnight. EcR–Usp complex (EcR) and supershifted EcR–Usp complex (ss) are indicated.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Incubation, Mutagenesis

dronc upstream promoter harbors a putative EcRBE. (A) In vitro translated EcR and Usp proteins were incubated with the EcR consensus probe hspEcRBE for 20 min in the presence of dronc promoter fragments. 400 bp PCR fragments spanning the dronc promoter region from 2.8 to 1.1 kb were gel purified and 400 ng was used in each reaction. Positive control ( hsp EcRBE) was used at 40 ng (equimolar). Complexes were resolved on an acrylamide/TBE gel, dried on 3 MM Whatmann paper, and exposed to Kodak film overnight. EcR–UsP complex is indicated. (B) EMSA experiment was performed as in A except PCR fragments used as competitors spanned the regions from 1.42 to 1.0 kb. hspEcRBE was used as the probe. (C) EMSA was performed as in A. Negative control competitor (D4) corresponds to the dronc promoter region between 67 to 7 bp upstream of the transcription start site. Oligonucleotide competitors correspond to the dronc promoter region 1.44 to 1.2 kb upstream of the transcription start site. Hsp EcRBE was used as the probe.
Figure Legend Snippet: dronc upstream promoter harbors a putative EcRBE. (A) In vitro translated EcR and Usp proteins were incubated with the EcR consensus probe hspEcRBE for 20 min in the presence of dronc promoter fragments. 400 bp PCR fragments spanning the dronc promoter region from 2.8 to 1.1 kb were gel purified and 400 ng was used in each reaction. Positive control ( hsp EcRBE) was used at 40 ng (equimolar). Complexes were resolved on an acrylamide/TBE gel, dried on 3 MM Whatmann paper, and exposed to Kodak film overnight. EcR–UsP complex is indicated. (B) EMSA experiment was performed as in A except PCR fragments used as competitors spanned the regions from 1.42 to 1.0 kb. hspEcRBE was used as the probe. (C) EMSA was performed as in A. Negative control competitor (D4) corresponds to the dronc promoter region between 67 to 7 bp upstream of the transcription start site. Oligonucleotide competitors correspond to the dronc promoter region 1.44 to 1.2 kb upstream of the transcription start site. Hsp EcRBE was used as the probe.

Techniques Used: In Vitro, Incubation, Polymerase Chain Reaction, Purification, Positive Control, Negative Control

Salivary glands and midgut express EcR-B1, which binds to dronc promoter. (A) Salivary glands and midgut were dissected from animals at −24, 2, or 12 h relative to puparium formation. RNA was analyzed by RT-PCR to detect EcR isoform expression. Rp49 was used as a control. (B) 9 μg of nuclear extracts prepared from various staged animals were incubated with the dronc EcRBE or the EcRBE mutant probe for 20 min. Complexes were analyzed as in Figs. 4 and 5 . EcR–Usp complex and supershift (ss) are indicated. Developmental stages are shown as hours relative to puparium formation. These stages represent early (0–4 h), mid (5–6 h), and late (9–11 h) prepupae and early (11–12 h) pupae. (C) EMSA was performed as in B in the presence of dronc EcRBE cold competitor, 2 μl of EcR common antibody, EcR-B1, or EcR-A. EcR–Usp (EcR) and supershifted EcR–Usp (ss) complexes are indicated. (D) EMSA was performed as in C with 6 μg on nuclear extract from salivary glands or midguts from 12 h (salivary gland) and 2 h (midgut) staged prepupae. EcR–Usp and supershifted (ss) complexes are shown. 40 ng of cold competitor ( dronc EcRBE) was also added where indicated.
Figure Legend Snippet: Salivary glands and midgut express EcR-B1, which binds to dronc promoter. (A) Salivary glands and midgut were dissected from animals at −24, 2, or 12 h relative to puparium formation. RNA was analyzed by RT-PCR to detect EcR isoform expression. Rp49 was used as a control. (B) 9 μg of nuclear extracts prepared from various staged animals were incubated with the dronc EcRBE or the EcRBE mutant probe for 20 min. Complexes were analyzed as in Figs. 4 and 5 . EcR–Usp complex and supershift (ss) are indicated. Developmental stages are shown as hours relative to puparium formation. These stages represent early (0–4 h), mid (5–6 h), and late (9–11 h) prepupae and early (11–12 h) pupae. (C) EMSA was performed as in B in the presence of dronc EcRBE cold competitor, 2 μl of EcR common antibody, EcR-B1, or EcR-A. EcR–Usp (EcR) and supershifted EcR–Usp (ss) complexes are indicated. (D) EMSA was performed as in C with 6 μg on nuclear extract from salivary glands or midguts from 12 h (salivary gland) and 2 h (midgut) staged prepupae. EcR–Usp and supershifted (ss) complexes are shown. 40 ng of cold competitor ( dronc EcRBE) was also added where indicated.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Mutagenesis

12) Product Images from "Production of p53 gene knockout rats by homologous recombination in embryonic stem cells"

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells

Journal: Nature

doi: 10.1038/nature09368

Confirmation of p53 gene targeting in rat ES cells a, Phase-contrast and fluorescence images of DAc8-p53-#1 rat ES cells. Scale bar, 50μm. b, Southern-blot analysis of p53 gene-targeted rat ES cells using 5′, 3′ or internal probes. For Southern blot analysis with 5′ or internal probes, genomic DNA from rat ES cells was digested with Spe I. For Southern blot analysis using 3′ probe, rat ES cell genomic DNA was digested with Hind III. The expected sizes of wild-type and p53 gene-targeted bands with different probes are shown in Fig. 1 . c, The diagram showing the positions of two PCR primer pairs. d-g, Sequence alignment of PCR products amplified from DAc8-p53-#1 rat ES cells. The sequence of the PCR product (Query) amplified using the first pair of primers (F1 and R1) was aligned with the sequences of the rat genome ( d ) or the p53 gene targeting vector ( e ). The sequence of the PCR product amplified using the second pair of primers (F2 and R2) was aligned with the sequences of the p53 gene targeting vector ( f ) or the rat genome ( g ). The highlighted sequences represent the junctions between the homology arms and the rat genome ( d and g ) or the CAG-EGFP-IRES-Pac cassette ( e and f ).
Figure Legend Snippet: Confirmation of p53 gene targeting in rat ES cells a, Phase-contrast and fluorescence images of DAc8-p53-#1 rat ES cells. Scale bar, 50μm. b, Southern-blot analysis of p53 gene-targeted rat ES cells using 5′, 3′ or internal probes. For Southern blot analysis with 5′ or internal probes, genomic DNA from rat ES cells was digested with Spe I. For Southern blot analysis using 3′ probe, rat ES cell genomic DNA was digested with Hind III. The expected sizes of wild-type and p53 gene-targeted bands with different probes are shown in Fig. 1 . c, The diagram showing the positions of two PCR primer pairs. d-g, Sequence alignment of PCR products amplified from DAc8-p53-#1 rat ES cells. The sequence of the PCR product (Query) amplified using the first pair of primers (F1 and R1) was aligned with the sequences of the rat genome ( d ) or the p53 gene targeting vector ( e ). The sequence of the PCR product amplified using the second pair of primers (F2 and R2) was aligned with the sequences of the p53 gene targeting vector ( f ) or the rat genome ( g ). The highlighted sequences represent the junctions between the homology arms and the rat genome ( d and g ) or the CAG-EGFP-IRES-Pac cassette ( e and f ).

Techniques Used: Fluorescence, Southern Blot, Polymerase Chain Reaction, Sequencing, Amplification, Plasmid Preparation

Germline transmission of the p53 gene-targeted mutation in the rat a, The male chimera generated from the DAc8-p53-#1 subclone. The agouti coat color and the appearance of GFP-positive tissues indicate that p53 gene-targeted DAc8 rat cells were present in the ES cell-rat chimera. b, Offspring produced by breeding the male chimera shown in ( a ) with SD female rats. c, Diagrams showing the positions of three PCR primers designed for genotyping p53 gene-targeted offspring. F1: gcg ttg ctc tga tgg tga c; F2: tgc ggt ggg ctc tat ggc ttc t ; R: cag cgt gat gat ggt aag gat. The expected sizes of PCR products for wild-type and p53 gene-targeted alleles are 309bp and 498bp, respectively. d, PCR genotyping analysis of the p53 gene-targeted allele. M, 100bp DNA marker; 1, DAc8 rat ES cells; 2, DAc8-p53-#1 rat ES cells; 3, 6, and 8, the three GFP-negative germline offspring; 4, 5, and 7, the three GFP-positive offspring; 9 and 10, the two albino littermates. e, Southern blot analysis for the p53 gene-targeted allele using the 5′ probe. 1, 4, and 6 are the three GFP-negative germline offspring; 2, 3, and 5 are the three GFP-positive offspring.
Figure Legend Snippet: Germline transmission of the p53 gene-targeted mutation in the rat a, The male chimera generated from the DAc8-p53-#1 subclone. The agouti coat color and the appearance of GFP-positive tissues indicate that p53 gene-targeted DAc8 rat cells were present in the ES cell-rat chimera. b, Offspring produced by breeding the male chimera shown in ( a ) with SD female rats. c, Diagrams showing the positions of three PCR primers designed for genotyping p53 gene-targeted offspring. F1: gcg ttg ctc tga tgg tga c; F2: tgc ggt ggg ctc tat ggc ttc t ; R: cag cgt gat gat ggt aag gat. The expected sizes of PCR products for wild-type and p53 gene-targeted alleles are 309bp and 498bp, respectively. d, PCR genotyping analysis of the p53 gene-targeted allele. M, 100bp DNA marker; 1, DAc8 rat ES cells; 2, DAc8-p53-#1 rat ES cells; 3, 6, and 8, the three GFP-negative germline offspring; 4, 5, and 7, the three GFP-positive offspring; 9 and 10, the two albino littermates. e, Southern blot analysis for the p53 gene-targeted allele using the 5′ probe. 1, 4, and 6 are the three GFP-negative germline offspring; 2, 3, and 5 are the three GFP-positive offspring.

Techniques Used: Transmission Assay, Mutagenesis, Generated, Produced, Polymerase Chain Reaction, Marker, Southern Blot

Generation of p53 gene knockout rats a, Offspring generated by intercrossing p53 tm1(EGFP-pac) heterozygote rats. b, Genotyping analysis on tail biopsies using PCR primers shown in Fig. 3c . M, 100bp DNA marker; 1–9, the nine GFP-positive offspring; 3 and 7, the two p53 tm1(EGFP-pac) homozygote pups; 10–12, the three GFP-negative offspring. c, Detection of p53 mRNA by Northern blot. Northern analysis was performed by sequential hybridization with probes for p53 and β-actin . d. Detection of p53 protein by Western blot.
Figure Legend Snippet: Generation of p53 gene knockout rats a, Offspring generated by intercrossing p53 tm1(EGFP-pac) heterozygote rats. b, Genotyping analysis on tail biopsies using PCR primers shown in Fig. 3c . M, 100bp DNA marker; 1–9, the nine GFP-positive offspring; 3 and 7, the two p53 tm1(EGFP-pac) homozygote pups; 10–12, the three GFP-negative offspring. c, Detection of p53 mRNA by Northern blot. Northern analysis was performed by sequential hybridization with probes for p53 and β-actin . d. Detection of p53 protein by Western blot.

Techniques Used: Gene Knockout, Generated, Polymerase Chain Reaction, Marker, Northern Blot, Hybridization, Western Blot

13) Product Images from "Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors"

Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors

Journal: Nature Communications

doi: 10.1038/s41467-017-02083-1

P. falciparum EV-DNA activates STING-dependent signaling in monocytes. a . THP-1 or STING KO THP-1 cells were incubated with P. falciparum ring-stage-derived or uRBC-derived vesicles for 1, 6 and 24 h. RT–PCR was performed for IFNA , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05. b . THP-1 or STING KO THP-1 cells were incubated with P. falciparum ring-stage or uRBC-derived vesicles for 1, 6 and 24 h. An ELISA assay was performed for CCL5 and CXCL10. HEK blue IFNα/β was performed. SD and T -test analysis * p ≤ 0.05. c . THP-1 cells were incubated with P. falciparum ring-stage-derived vesicles, P. falciparum gDNA or transfected with poly(dA:dT) for 24 h. WB analysis was performed for STING, pIRF3, pTBK1 and α actin, sm-size marker. d . THP-1 cells were incubated with P. falciparum ring stage-derived vesicles for 24 h. Confocal microscopy images were taken for STING, pIRF3, pTBK1 (FITC), and DAPI. Scale bar 10 μm
Figure Legend Snippet: P. falciparum EV-DNA activates STING-dependent signaling in monocytes. a . THP-1 or STING KO THP-1 cells were incubated with P. falciparum ring-stage-derived or uRBC-derived vesicles for 1, 6 and 24 h. RT–PCR was performed for IFNA , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05. b . THP-1 or STING KO THP-1 cells were incubated with P. falciparum ring-stage or uRBC-derived vesicles for 1, 6 and 24 h. An ELISA assay was performed for CCL5 and CXCL10. HEK blue IFNα/β was performed. SD and T -test analysis * p ≤ 0.05. c . THP-1 cells were incubated with P. falciparum ring-stage-derived vesicles, P. falciparum gDNA or transfected with poly(dA:dT) for 24 h. WB analysis was performed for STING, pIRF3, pTBK1 and α actin, sm-size marker. d . THP-1 cells were incubated with P. falciparum ring stage-derived vesicles for 24 h. Confocal microscopy images were taken for STING, pIRF3, pTBK1 (FITC), and DAPI. Scale bar 10 μm

Techniques Used: Incubation, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Marker, Confocal Microscopy

P. falciparum DNA stimulates innate immune gene induction in monocytes. a THP-1 cells were transfected or incubated with P. falciparum genomic DNA for 1, 6, and 24 h. RT–PCR was performed for the products IFNα , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05, ** p ≤ 0.01. b THP-1 cells were transfected or incubated with poly(dA:dT) for 1, 6, and 24 h. RT–PCR was performed for IFNA , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05, ** p ≤ 0.01. c THP-1 cells were transfected or incubated with P. falciparum genomic DNA for 1, 6, and 24 h. ELISA were performed for CXCL10 and CCL5 and HEK blue IFNα/β. SD and T -test analysis * p ≤ 0.05. d THP-1 cells were transfected or incubated with poly(dA:dT) for 1, 6, and 24 h. ELISA were performed for CXCL10 and CCL5 and HEK blue IFNα/β. SD and T -test analysis * p ≤ 0.05
Figure Legend Snippet: P. falciparum DNA stimulates innate immune gene induction in monocytes. a THP-1 cells were transfected or incubated with P. falciparum genomic DNA for 1, 6, and 24 h. RT–PCR was performed for the products IFNα , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05, ** p ≤ 0.01. b THP-1 cells were transfected or incubated with poly(dA:dT) for 1, 6, and 24 h. RT–PCR was performed for IFNA , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05, ** p ≤ 0.01. c THP-1 cells were transfected or incubated with P. falciparum genomic DNA for 1, 6, and 24 h. ELISA were performed for CXCL10 and CCL5 and HEK blue IFNα/β. SD and T -test analysis * p ≤ 0.05. d THP-1 cells were transfected or incubated with poly(dA:dT) for 1, 6, and 24 h. ELISA were performed for CXCL10 and CCL5 and HEK blue IFNα/β. SD and T -test analysis * p ≤ 0.05

Techniques Used: Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

DNA-binding proteins and mitochondrial and apicoplast genes in EVs. a PCR for Ev-DNA markers, apicoplast ( ssu-api ), mitochondria ( ssud ) and nuclear genes ( msp2 , rap14 , and gap40 ). Plasmid control PuF-1 was added externally to EVs prior to DNase I treatment. b P. falciparum msp2 gene Ev-FISH images of ring and trophozoite EVs and uRBC vesicles. c P. falciparum ssu-api and ssud genes Ev-FISH of ring EVs. d H3 and H4 protein WB analysis for 2–5 OP gradient fractions (F). e P. falciparum H4 protein IFA. f P . falciparum parasites release EVs during the early post-invasion phase. Fluorescence microscopy using DAPI in EVs produced by iRBCs across their life cycle. Images of EVs collected at 12, 24, 36 and 48 h post invasion. Giemsa stains (first column) show the state of the parasites prior to collecting EVs at each time point. g SR1 control IFA
Figure Legend Snippet: DNA-binding proteins and mitochondrial and apicoplast genes in EVs. a PCR for Ev-DNA markers, apicoplast ( ssu-api ), mitochondria ( ssud ) and nuclear genes ( msp2 , rap14 , and gap40 ). Plasmid control PuF-1 was added externally to EVs prior to DNase I treatment. b P. falciparum msp2 gene Ev-FISH images of ring and trophozoite EVs and uRBC vesicles. c P. falciparum ssu-api and ssud genes Ev-FISH of ring EVs. d H3 and H4 protein WB analysis for 2–5 OP gradient fractions (F). e P. falciparum H4 protein IFA. f P . falciparum parasites release EVs during the early post-invasion phase. Fluorescence microscopy using DAPI in EVs produced by iRBCs across their life cycle. Images of EVs collected at 12, 24, 36 and 48 h post invasion. Giemsa stains (first column) show the state of the parasites prior to collecting EVs at each time point. g SR1 control IFA

Techniques Used: DNA Binding Assay, Polymerase Chain Reaction, Plasmid Preparation, Fluorescence In Situ Hybridization, Western Blot, Immunofluorescence, Fluorescence, Microscopy, Produced

14) Product Images from "The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A"

Article Title: The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

Journal: Toxicology and applied pharmacology

doi: 10.1016/j.taap.2013.08.017

Human MT2A expression is induced by AHR agonists in human cultured cells. (a, b) HepG2 cells were treated with TCDD (a) or 3-MC (b) at the indicated concentrations for 9 h, and MT2A mRNA levels were determined using quantitative RT-PCR. Controls were treated with the vehicle (−). (c) HeLa cells were transfected with 0.25 μg of pMT2A-Luc and 0.15 μg of pGL4-hRluc-TK together with either 0.1 μg of AHR expression plasmid or control plasmid for 3 h. Cells were treated for 24 h with 1 μM 3-MC or vehicle, and relative luciferase activities were determined. The values are the mean ± SD of 3 (a, b) or 2 (c) independent experiments. Values with an asterisk are significantly different from vehicle control at p
Figure Legend Snippet: Human MT2A expression is induced by AHR agonists in human cultured cells. (a, b) HepG2 cells were treated with TCDD (a) or 3-MC (b) at the indicated concentrations for 9 h, and MT2A mRNA levels were determined using quantitative RT-PCR. Controls were treated with the vehicle (−). (c) HeLa cells were transfected with 0.25 μg of pMT2A-Luc and 0.15 μg of pGL4-hRluc-TK together with either 0.1 μg of AHR expression plasmid or control plasmid for 3 h. Cells were treated for 24 h with 1 μM 3-MC or vehicle, and relative luciferase activities were determined. The values are the mean ± SD of 3 (a, b) or 2 (c) independent experiments. Values with an asterisk are significantly different from vehicle control at p

Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase

Dex and AHR agonists increase MT2A expression. HepG2 cells were treated for 9 h with vehicle, 10 nM TCDD and/or 100 nM Dex (a, b) or 1 μM 3-MC and/or 100 nM Dex (c). The mRNA levels were determined using quantitative RT-PCR. The protein levels of AHR and GR were determined by Western blotting (c, lower panel). (d) HepG2 cells were treated for 9 h with 3-MC and/or Dex, or vehicle at the indicated concentrations. MT2A mRNA levels were determined using quantitative RT-PCR. (e) HeLa cells were transfected with 0.25 μg of pMT2A-Luc and 0.15 μg of pGL4-hRluc-TK for 3 h. Cells were treated for 24 h with 1 μM 3-MC and/or 10 nM Dex, or vehicle, and relative luciferase activities were determined. The values are the mean ± SD of 3 (a, b, c, e) or 2 (d) independent experiments. Values with an asterisk are significantly different compared to vehicle control; the value with a sharp was significantly different compared to treatment with either 3-MC or Dex at p
Figure Legend Snippet: Dex and AHR agonists increase MT2A expression. HepG2 cells were treated for 9 h with vehicle, 10 nM TCDD and/or 100 nM Dex (a, b) or 1 μM 3-MC and/or 100 nM Dex (c). The mRNA levels were determined using quantitative RT-PCR. The protein levels of AHR and GR were determined by Western blotting (c, lower panel). (d) HepG2 cells were treated for 9 h with 3-MC and/or Dex, or vehicle at the indicated concentrations. MT2A mRNA levels were determined using quantitative RT-PCR. (e) HeLa cells were transfected with 0.25 μg of pMT2A-Luc and 0.15 μg of pGL4-hRluc-TK for 3 h. Cells were treated for 24 h with 1 μM 3-MC and/or 10 nM Dex, or vehicle, and relative luciferase activities were determined. The values are the mean ± SD of 3 (a, b, c, e) or 2 (d) independent experiments. Values with an asterisk are significantly different compared to vehicle control; the value with a sharp was significantly different compared to treatment with either 3-MC or Dex at p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase

AHR interacts with GR. (a) HepG2 cells were treated for 30 min with 1 μM 3-MC and 100 nM Dex, or control vehicle, and ChIP assays were performed. The levels of the PCR products for the EEF1A1 exon, GRE and XRE were normalized to the control IgG. The fold enrichment of GRE or XRE is presented as a ratio to the EEF1A1 exon expression. Independent experiments were performed 3 times. (b) COS7 cells were transfected with 0.25 μg of the GAL4 response element-driven luciferase reporter plasmid and 0.15 μg of the pGL4-hRluc-TK construct together with either 0.1 μg of the expression plasmids containing the full-length receptor fusion sequence (pM-AHR and/or pVP16-GR) or the control plasmids (pM or pVP16) for 24 h. Cells were lysed and relative luciferase activities were determined. Values are the mean ± SD of 3 independent experiments. The value with asterisk is significantly different compared to control at p
Figure Legend Snippet: AHR interacts with GR. (a) HepG2 cells were treated for 30 min with 1 μM 3-MC and 100 nM Dex, or control vehicle, and ChIP assays were performed. The levels of the PCR products for the EEF1A1 exon, GRE and XRE were normalized to the control IgG. The fold enrichment of GRE or XRE is presented as a ratio to the EEF1A1 exon expression. Independent experiments were performed 3 times. (b) COS7 cells were transfected with 0.25 μg of the GAL4 response element-driven luciferase reporter plasmid and 0.15 μg of the pGL4-hRluc-TK construct together with either 0.1 μg of the expression plasmids containing the full-length receptor fusion sequence (pM-AHR and/or pVP16-GR) or the control plasmids (pM or pVP16) for 24 h. Cells were lysed and relative luciferase activities were determined. Values are the mean ± SD of 3 independent experiments. The value with asterisk is significantly different compared to control at p

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Plasmid Preparation, Construct, Sequencing

15) Product Images from "Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment"

Article Title: Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment

Journal: PLoS ONE

doi: 10.1371/journal.pone.0104631

Grsf1 controls translation of Use1. ( A ) Luciferase reporter constructs harboring the Use1 wt 5′UTR (WT, black bars), or a 5′UTR lacking the AGGGCGGA repeat (open bars), were transfected in NIH3T3 cells together with increasing amounts of a Grsf1 expression plasmid. The total amount of transfected DNA was kept constant by adding additional backbone plasmid DNA (EV, pcDNA3.1). Luciferase activity is corrected for mRNA expression and given as fold-change (fc) compared to WT reporter in absence of Grsf1 overexpression. Error bars indicate standard deviation of 3 experiments. ( B ) I/11cells were transduced with the lentiviral constructs containing two different shRNAs matching Grsf1 (indicated a and b) or control shRNA (Scr). The efficiency of Grsf1 transcript knock down was determined using RT-PCR. The expression of Grsf1 in parental cells is set to 1. Error bars indicate standard deviation of 3 experiments. ( C ) The protein lysates from transduced I/11 cells (representative experiment from B) were tested in WB with anti-actin (42 kDa) and anti-Use1 (30.5 kDa) antibody.
Figure Legend Snippet: Grsf1 controls translation of Use1. ( A ) Luciferase reporter constructs harboring the Use1 wt 5′UTR (WT, black bars), or a 5′UTR lacking the AGGGCGGA repeat (open bars), were transfected in NIH3T3 cells together with increasing amounts of a Grsf1 expression plasmid. The total amount of transfected DNA was kept constant by adding additional backbone plasmid DNA (EV, pcDNA3.1). Luciferase activity is corrected for mRNA expression and given as fold-change (fc) compared to WT reporter in absence of Grsf1 overexpression. Error bars indicate standard deviation of 3 experiments. ( B ) I/11cells were transduced with the lentiviral constructs containing two different shRNAs matching Grsf1 (indicated a and b) or control shRNA (Scr). The efficiency of Grsf1 transcript knock down was determined using RT-PCR. The expression of Grsf1 in parental cells is set to 1. Error bars indicate standard deviation of 3 experiments. ( C ) The protein lysates from transduced I/11 cells (representative experiment from B) were tested in WB with anti-actin (42 kDa) and anti-Use1 (30.5 kDa) antibody.

Techniques Used: Luciferase, Construct, Transfection, Expressing, Plasmid Preparation, Activity Assay, Over Expression, Standard Deviation, Transduction, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot

The unspliced transcript is preferentially translated. ( A ) Polysome profile of I/ll cells. Cell lysate was centrifuged on a 7–46% sucrose gradient and the distribution of RNA was measured by absorption at 254 nm. The free mRNA, ribosomal subunits and polysomes are indicated. ( B ) Real time, reverse transcriptase PCR on polysome bound (pb) and subpolysomal (sub) mRNA using a reverse primer downstream the Use1 AUG start codon and forward primers upstream of, and within the intron to amplify the spliced (black bars) and unspliced (white bars) RNA, respectively. The percentage polysome recruitment was calculated for both transcripts. ( C ) The spliced mRNA variant encodes a longer protein variant. The top nucleotide sequence represents the unspliced transcript. At −154 and +3 the AG nucleotides that are part of the splice sites are shown in capitals. The lower nucleotide sequence represents the spliced transcript. A start codon at nucleotide −108, compliant with the Kazak consensus, appears to be in frame with the main ORF in the spliced transcript, whereas it is followed by stop codon in the intron in the unspliced transcript. Predicted protein sequences are shown above the nucleotide sequences. Slashes indicate gaps in the sequence as only important parts of the sequence are shown. ( D ) Protein expression in cultured I/11 erythroblasts and in primary liver cells (C57/B6). Western blot probed with anti-Use1, and with anti-Stat3 as loading control. Use 1 isoforms are indicated.
Figure Legend Snippet: The unspliced transcript is preferentially translated. ( A ) Polysome profile of I/ll cells. Cell lysate was centrifuged on a 7–46% sucrose gradient and the distribution of RNA was measured by absorption at 254 nm. The free mRNA, ribosomal subunits and polysomes are indicated. ( B ) Real time, reverse transcriptase PCR on polysome bound (pb) and subpolysomal (sub) mRNA using a reverse primer downstream the Use1 AUG start codon and forward primers upstream of, and within the intron to amplify the spliced (black bars) and unspliced (white bars) RNA, respectively. The percentage polysome recruitment was calculated for both transcripts. ( C ) The spliced mRNA variant encodes a longer protein variant. The top nucleotide sequence represents the unspliced transcript. At −154 and +3 the AG nucleotides that are part of the splice sites are shown in capitals. The lower nucleotide sequence represents the spliced transcript. A start codon at nucleotide −108, compliant with the Kazak consensus, appears to be in frame with the main ORF in the spliced transcript, whereas it is followed by stop codon in the intron in the unspliced transcript. Predicted protein sequences are shown above the nucleotide sequences. Slashes indicate gaps in the sequence as only important parts of the sequence are shown. ( D ) Protein expression in cultured I/11 erythroblasts and in primary liver cells (C57/B6). Western blot probed with anti-Use1, and with anti-Stat3 as loading control. Use 1 isoforms are indicated.

Techniques Used: Polymerase Chain Reaction, Variant Assay, Sequencing, Expressing, Cell Culture, Western Blot

Reduced expression of Use1 or Grsf1 abrogates expansion of erythroblasts. Erythroblasts (I/11 cell line) were transduced with lentiviral shRNA vectors repressing expression of Use1 (2 distinct sequences, indicated a and b) or Grsf1 (2 distinct sequences, indicated a and b). Panels show representative data from 1 of 4 replicates. ( A ) Use1 RNA expression was controlled by RT-PCR 4 days following transfection. For Grsf1 knockdown see figure 4B ) ( B ) Cytospins of the cultured cells made 4 days post transduction showed mainly blasts when expressing control shRNA (Scr). Expression of shRNAs matching Use1 or Grsf1 increased the number of more mature hemoglobinised cells (Dark staining, white arrows) and pycnotic cells (grey arrows). A quantification of cell types, based on counting > 300 cells, is shown above the cytospin as pie-diagram (black, blasts; white, maturing red cells; grey, pycnotic cells). ( C ) shRNA treated cells were counted daily, kept at 2×10 6 /ml and cumulative cell numbers were calculated. ( D ) At the start and 96 hours following induction of differentiation the hemoglobin concentration was measured and calculated as Hb/cell volume in arbitrary units (A.U.). ( E ) At 96 hours the ratio of life/dead cells was determined by staining with propidium iodide (PI). Cells positive for PI were measured by flowcytometry (LSRII, BD).
Figure Legend Snippet: Reduced expression of Use1 or Grsf1 abrogates expansion of erythroblasts. Erythroblasts (I/11 cell line) were transduced with lentiviral shRNA vectors repressing expression of Use1 (2 distinct sequences, indicated a and b) or Grsf1 (2 distinct sequences, indicated a and b). Panels show representative data from 1 of 4 replicates. ( A ) Use1 RNA expression was controlled by RT-PCR 4 days following transfection. For Grsf1 knockdown see figure 4B ) ( B ) Cytospins of the cultured cells made 4 days post transduction showed mainly blasts when expressing control shRNA (Scr). Expression of shRNAs matching Use1 or Grsf1 increased the number of more mature hemoglobinised cells (Dark staining, white arrows) and pycnotic cells (grey arrows). A quantification of cell types, based on counting > 300 cells, is shown above the cytospin as pie-diagram (black, blasts; white, maturing red cells; grey, pycnotic cells). ( C ) shRNA treated cells were counted daily, kept at 2×10 6 /ml and cumulative cell numbers were calculated. ( D ) At the start and 96 hours following induction of differentiation the hemoglobin concentration was measured and calculated as Hb/cell volume in arbitrary units (A.U.). ( E ) At 96 hours the ratio of life/dead cells was determined by staining with propidium iodide (PI). Cells positive for PI were measured by flowcytometry (LSRII, BD).

Techniques Used: Expressing, Transduction, shRNA, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Transfection, Cell Culture, Staining, Concentration Assay

16) Product Images from "p75 neurotrophin receptor mediates apoptosis in transit-amplifying cells and its overexpression restores cell death in psoriatic keratinocytes"

Article Title: p75 neurotrophin receptor mediates apoptosis in transit-amplifying cells and its overexpression restores cell death in psoriatic keratinocytes

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2010.162

Pro-NGF mediates apoptosis in human keratinocytes through p75NTR and sortilin. ( a ) Normal human keratinocytes were obtained from neonatal foreskin. A real-time PCR was performed on RNA extracts by using primers for p75NTR and sortilin, as described in Materials and Methods section. p75NTR expression levels were used as calibrator. ( b ) Keratinocyte cultures were trypsinized and stained with mouse monoclonal anti-p75NTR and polyclonal anti-sortilin antibody. Cells were analyzed by flow cytometry. ( c ) Normal human keratinocytes were separated into three sub-populations on type IV collagen. Real-time PCR was performed on RNA extracts of the three populations by using primers for sortilin, as described in Materials and Methods section. KSCs were used as calibrator. ( d ) Protein extracts from the three populations were separated on 7% polyacrylamide gels and transferred onto nitrocellulose membrane. Membranes were immunoblotted with anti-sortilin antibody. ( e ) Keratinocyte cultures, seeded in a 96-well tissue culture plate, were treated with 0.1, 1, 10, 20 ng/ml of pro-NGF. MTT assay was performed after 72 h. ( f ) Keratinocyte confluent cultures, seeded in a 96-wells tissue culture plate, were treated with 10 ng/ml of pro-NGF or 200 nM k252a alone, or in combination. MTT assay was performed after 72 h. ( g ) Confluent keratinocytes, seeded in a tissue culture slide-flask, were treated as described above. At 72 h, cells were fixed and stained in situ with TUNEL. Positive cells were counted as described in Materials and Methods section. ( h ) HaCaT cells were transfected with 2 μ g of pcDNA3-BimEL (positive control) in combination with 2 μ g of pcDNA3-p75NTR and pcDNA3-sortilin. Transfection was controlled by western blotting using anti-sortilin and anti-p75NTR antibody. ( i ) After 24 h, cells were treated with 10 ng/ml of pro-NGF for 24 h. Thereafter, HaCaT cells were lysed and caspase activity was assessed using DEVD-AFC substrate. Data represent the mean±S.E. of triplicate determinations
Figure Legend Snippet: Pro-NGF mediates apoptosis in human keratinocytes through p75NTR and sortilin. ( a ) Normal human keratinocytes were obtained from neonatal foreskin. A real-time PCR was performed on RNA extracts by using primers for p75NTR and sortilin, as described in Materials and Methods section. p75NTR expression levels were used as calibrator. ( b ) Keratinocyte cultures were trypsinized and stained with mouse monoclonal anti-p75NTR and polyclonal anti-sortilin antibody. Cells were analyzed by flow cytometry. ( c ) Normal human keratinocytes were separated into three sub-populations on type IV collagen. Real-time PCR was performed on RNA extracts of the three populations by using primers for sortilin, as described in Materials and Methods section. KSCs were used as calibrator. ( d ) Protein extracts from the three populations were separated on 7% polyacrylamide gels and transferred onto nitrocellulose membrane. Membranes were immunoblotted with anti-sortilin antibody. ( e ) Keratinocyte cultures, seeded in a 96-well tissue culture plate, were treated with 0.1, 1, 10, 20 ng/ml of pro-NGF. MTT assay was performed after 72 h. ( f ) Keratinocyte confluent cultures, seeded in a 96-wells tissue culture plate, were treated with 10 ng/ml of pro-NGF or 200 nM k252a alone, or in combination. MTT assay was performed after 72 h. ( g ) Confluent keratinocytes, seeded in a tissue culture slide-flask, were treated as described above. At 72 h, cells were fixed and stained in situ with TUNEL. Positive cells were counted as described in Materials and Methods section. ( h ) HaCaT cells were transfected with 2 μ g of pcDNA3-BimEL (positive control) in combination with 2 μ g of pcDNA3-p75NTR and pcDNA3-sortilin. Transfection was controlled by western blotting using anti-sortilin and anti-p75NTR antibody. ( i ) After 24 h, cells were treated with 10 ng/ml of pro-NGF for 24 h. Thereafter, HaCaT cells were lysed and caspase activity was assessed using DEVD-AFC substrate. Data represent the mean±S.E. of triplicate determinations

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Staining, Flow Cytometry, Cytometry, MTT Assay, In Situ, TUNEL Assay, Transfection, Positive Control, Western Blot, Activity Assay

p75NTR-induced apoptosis in TA cells. ( a ) Cryosections of normal skin were acetone-fixed and stained with anti-p75NTR antibody. Fast red was used as cromogen for p75NTR. Cryosections were double stained with anti-p75NTR and anti-keratin 10 (CK10) antibodies or with anti-p75NTR and transglutaminase type I (TgaseI) antibodies. Fast blue was used as cromogen for p75NTR, whereas carbazol for CK10 and TGaseI. ( b ) Normal human keratinocytes were obtained from neonatal foreskin and separated into three sub-populations on type IV collagen. A real-time PCR was performed on RNA extracts of the three populations by using primers for p75NTR, as described in Materials and Methods section. KSC were used as calibrator. Student's t -test was performed between samples and calibrator. ( c ) Protein extracts from the three populations were separated on 7% polyacrylamide gels and transferred onto nitrocellulose membrane. Membranes were immunoblotted with anti-p75NTR antibody. β -actin was used as internal control. ( d ) KSC and TA cells were treated with either BDNF or β -amyloid, stained with PI, and analyzed by flow cytometry. ( e ) TA cells were transiently transfected with 50 nM of p75NTR siRNA, as shown by western blotting. ( f ) At 48 h after transfection, both mock and transfected cells were treated with BDNF (100 ng/ml) or β -amyloid (40 μ M). SubG1 peak analysis was performed after 24 h
Figure Legend Snippet: p75NTR-induced apoptosis in TA cells. ( a ) Cryosections of normal skin were acetone-fixed and stained with anti-p75NTR antibody. Fast red was used as cromogen for p75NTR. Cryosections were double stained with anti-p75NTR and anti-keratin 10 (CK10) antibodies or with anti-p75NTR and transglutaminase type I (TgaseI) antibodies. Fast blue was used as cromogen for p75NTR, whereas carbazol for CK10 and TGaseI. ( b ) Normal human keratinocytes were obtained from neonatal foreskin and separated into three sub-populations on type IV collagen. A real-time PCR was performed on RNA extracts of the three populations by using primers for p75NTR, as described in Materials and Methods section. KSC were used as calibrator. Student's t -test was performed between samples and calibrator. ( c ) Protein extracts from the three populations were separated on 7% polyacrylamide gels and transferred onto nitrocellulose membrane. Membranes were immunoblotted with anti-p75NTR antibody. β -actin was used as internal control. ( d ) KSC and TA cells were treated with either BDNF or β -amyloid, stained with PI, and analyzed by flow cytometry. ( e ) TA cells were transiently transfected with 50 nM of p75NTR siRNA, as shown by western blotting. ( f ) At 48 h after transfection, both mock and transfected cells were treated with BDNF (100 ng/ml) or β -amyloid (40 μ M). SubG1 peak analysis was performed after 24 h

Techniques Used: Staining, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Transfection, Western Blot

17) Product Images from "Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat"

Article Title: Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat

Journal: Plant Methods

doi: 10.1186/1746-4811-6-26

Silencing of HvIPS1 in barley roots and shoots . A HvIPS1 RNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants inoculated with BSMV-IPS1 (black bars) or BSMV-GFP 250 (white bars). Plants were grown in 0 (5, 7, 9 dpi) or 1 mM Pi (9 dpi) and harvested at 5, 7, or 9 dpi. Each bar represents three samples (with one exception - only two samples for the 0 Pi, GFP, 9dpi bar). Error bars denote standard deviations. AU - Arbitrary units. B Stability of the BSMV-IPS1 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8, 9: 0 mM Pi, 9 dpi; 10, 11, 12: 1 mM Pi, 9 dpi; 13: plasmid containing BSMVγ-IPS1; 14: water control. M4: GeneRuler 50 bp DNA Ladder (Fermentas); white arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. C Stability of the BSMV-GFP 250 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8: 0 mM Pi, 9 dpi; 9, 10, 11: 1 mM Pi, 9 dpi. D HvIPS1 RNA expression levels in root and shoot tissue determined by qRT-PCR (normalization to ubiquitin). BSMV-IPS1 (black bars), BSMV-GFP 250 (white bars). Plant were grown in 0 mM Pi and harvested at 9 dpi. Each bar represents five samples. Error bars denote standard deviations.
Figure Legend Snippet: Silencing of HvIPS1 in barley roots and shoots . A HvIPS1 RNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants inoculated with BSMV-IPS1 (black bars) or BSMV-GFP 250 (white bars). Plants were grown in 0 (5, 7, 9 dpi) or 1 mM Pi (9 dpi) and harvested at 5, 7, or 9 dpi. Each bar represents three samples (with one exception - only two samples for the 0 Pi, GFP, 9dpi bar). Error bars denote standard deviations. AU - Arbitrary units. B Stability of the BSMV-IPS1 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8, 9: 0 mM Pi, 9 dpi; 10, 11, 12: 1 mM Pi, 9 dpi; 13: plasmid containing BSMVγ-IPS1; 14: water control. M4: GeneRuler 50 bp DNA Ladder (Fermentas); white arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. C Stability of the BSMV-GFP 250 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8: 0 mM Pi, 9 dpi; 9, 10, 11: 1 mM Pi, 9 dpi. D HvIPS1 RNA expression levels in root and shoot tissue determined by qRT-PCR (normalization to ubiquitin). BSMV-IPS1 (black bars), BSMV-GFP 250 (white bars). Plant were grown in 0 mM Pi and harvested at 9 dpi. Each bar represents five samples. Error bars denote standard deviations.

Techniques Used: RNA Expression, Quantitative RT-PCR, Construct, Polymerase Chain Reaction, Plasmid Preparation

BSMVγ constructs selected for relative stability also produce successful gene silencing in barley roots . A RT-PCR analysis of the stability of BSMVγ constructs in barley roots. Expected lengths for the PCR products are presented in brackets. Lanes 1, 2: BSMV-IPS1 (493 bp); 3, 4: BSMV-Pht1;1 (610 bp); 5, 6: BSMV-Pht1;4 (616 bp); 7, 8: BSMV-Pht1;7 (623 bp); 9, 10: BSMV-PHR1 (495 bp); 11, 12: BSMV-PHO2 247 (511 bp); 13, 14: BSMV-PHO2 387 (651 bp); 15, 16: GFP 250 (492 bp); 17: control plasmid carrying BSMVγ-GFP 375 (617 bp); 18: control plasmid carrying BSMVγ-IPS1 (493 bp); M4: GeneRuler 50 bp DNA Ladder (Fermentas); M5: O'GeneRuler 50bp DNA Ladder (Fermentas). White arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. B and C HvPHR1 expression levels in root tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHR1 (black bars), BSMV-GFP 250 (white bars). Samples in B are identical to those shown in A, lanes 9, 10 and 15,16. In C, each bar represents five independent samples. AU - arbitrary units; error bars denote standard deviations. D HvPHO2 expression levels in root and leaf tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHO2 247 (black bars), BSMV-GFP 250 (white bars). E As D, but bars represent Pi content in inoculated plants (in μmol Pi/g of fresh weight).
Figure Legend Snippet: BSMVγ constructs selected for relative stability also produce successful gene silencing in barley roots . A RT-PCR analysis of the stability of BSMVγ constructs in barley roots. Expected lengths for the PCR products are presented in brackets. Lanes 1, 2: BSMV-IPS1 (493 bp); 3, 4: BSMV-Pht1;1 (610 bp); 5, 6: BSMV-Pht1;4 (616 bp); 7, 8: BSMV-Pht1;7 (623 bp); 9, 10: BSMV-PHR1 (495 bp); 11, 12: BSMV-PHO2 247 (511 bp); 13, 14: BSMV-PHO2 387 (651 bp); 15, 16: GFP 250 (492 bp); 17: control plasmid carrying BSMVγ-GFP 375 (617 bp); 18: control plasmid carrying BSMVγ-IPS1 (493 bp); M4: GeneRuler 50 bp DNA Ladder (Fermentas); M5: O'GeneRuler 50bp DNA Ladder (Fermentas). White arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. B and C HvPHR1 expression levels in root tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHR1 (black bars), BSMV-GFP 250 (white bars). Samples in B are identical to those shown in A, lanes 9, 10 and 15,16. In C, each bar represents five independent samples. AU - arbitrary units; error bars denote standard deviations. D HvPHO2 expression levels in root and leaf tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHO2 247 (black bars), BSMV-GFP 250 (white bars). E As D, but bars represent Pi content in inoculated plants (in μmol Pi/g of fresh weight).

Techniques Used: Construct, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation, Expressing, Quantitative RT-PCR

Attempt at silencing HvPht1;1 expression in barley roots . A HvPht1;1 mRNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants were inoculated with either BSMV-Pht1;1 (black bars) or BSMV-GFP 375 (white bars). Plants were grown in hydroponic culture containing 0 or 1 mM Pi. Each bar represents three samples, error bars denote standard deviations. AU - Arbitrary units. B Schematic view of BSMVγ with introduced insert. Arrows show the position of the primers BSMVgbF and BSMVgbR used for assessing stability of insert. The length of virus sequence 5' and 3' of the insert is shown. PCR products of
Figure Legend Snippet: Attempt at silencing HvPht1;1 expression in barley roots . A HvPht1;1 mRNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants were inoculated with either BSMV-Pht1;1 (black bars) or BSMV-GFP 375 (white bars). Plants were grown in hydroponic culture containing 0 or 1 mM Pi. Each bar represents three samples, error bars denote standard deviations. AU - Arbitrary units. B Schematic view of BSMVγ with introduced insert. Arrows show the position of the primers BSMVgbF and BSMVgbR used for assessing stability of insert. The length of virus sequence 5' and 3' of the insert is shown. PCR products of "USER cloning" BSMV vectors are longer by 22 bp compared with restriction enzymes cloning. Not drawn to scale. C Stability of the BSMV-Pht1;1 and BSMV-GFP 375 constructs in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert as shown in B. Lanes 1, 2, 3: BSMV-Pht1;1, 0 mM Pi (610 bp); 4, 5, 6: BSMV-Pht1;1, 1 mM Pi (610 bp); 7, 8, 9: BSMV-GFP 375 , 0 mM Pi (617 bp); 10, 11, 12: BSMV-GFP 375 ,1 mM Pi (617 bp); 13: plasmid containing BSMVγ-PDS cassette (643 bp); 14: water control; M2: DNA marker; black arrow represents DNA fragment of 564 bp. The expected lengths for PCR products are given in brackets.

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Polymerase Chain Reaction, Clone Assay, Construct, Plasmid Preparation, Marker

18) Product Images from "Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection"

Article Title: Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp604

ADAR1 associates with HIV-1 RNAs. 293T cells were transiently co-transfected with miniB13 plasmid (2 μg) and pEGFP-ADAR1 (8 μg) in the presence or absence of NL4-3 proviral DNA (15 μg). Forty-eight hours post-transfection total cell extracts were prepared and immunoprecipitated with anti-ADAR1 antibody or with control rabbit IgGs. ( A ) Western blotting analysis of an aliquot of the immunoprecipitated proteins and total cell extract probed with anti-ADAR1 antibody. ( B ) RNA isolated from the immunoprecipitates was retrotranscribed and amplified by PCR using specific primers to detect the TAR sequence harbored at the extreme 5′ end of all HIV-1 transcripts, the miniB13 transcript and the endogenous GAPDH mRNA. As a positive control, an RT–PCR experiment was performed with total RNA isolated from 293T co-transfected with miniB13 plasmid along with pEGFP-ADAR1, and NL4-3 proviral DNA (Cell ext.). Exclusion of RT from the RT–PCR reaction served as a specific negative control. ( C ) An RT–PCR analysis was carried using specific primers designed to distinguish the different HIV-1 RNA species (unspliced 9-kb RNA, spliced 4-kb and 2-kb RNAs) associated with ADAR1.
Figure Legend Snippet: ADAR1 associates with HIV-1 RNAs. 293T cells were transiently co-transfected with miniB13 plasmid (2 μg) and pEGFP-ADAR1 (8 μg) in the presence or absence of NL4-3 proviral DNA (15 μg). Forty-eight hours post-transfection total cell extracts were prepared and immunoprecipitated with anti-ADAR1 antibody or with control rabbit IgGs. ( A ) Western blotting analysis of an aliquot of the immunoprecipitated proteins and total cell extract probed with anti-ADAR1 antibody. ( B ) RNA isolated from the immunoprecipitates was retrotranscribed and amplified by PCR using specific primers to detect the TAR sequence harbored at the extreme 5′ end of all HIV-1 transcripts, the miniB13 transcript and the endogenous GAPDH mRNA. As a positive control, an RT–PCR experiment was performed with total RNA isolated from 293T co-transfected with miniB13 plasmid along with pEGFP-ADAR1, and NL4-3 proviral DNA (Cell ext.). Exclusion of RT from the RT–PCR reaction served as a specific negative control. ( C ) An RT–PCR analysis was carried using specific primers designed to distinguish the different HIV-1 RNA species (unspliced 9-kb RNA, spliced 4-kb and 2-kb RNAs) associated with ADAR1.

Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Isolation, Amplification, Polymerase Chain Reaction, Sequencing, Positive Control, Reverse Transcription Polymerase Chain Reaction, Negative Control

A-to-I RNA editing at specific sites in the Rev and Tat coding sequence. The Rev and Tat coding sequence were analyzed by direct sequencing of RT–PCR products generated using as substrate the total RNA isolated as described in Figure 6 . Three independent co-transfection experiments with relative RT–PCR analysis were carried out. The edited adenosines found in the Rev and Tat coding sequence are numbered (relative to NL4-3) and shown in bold. The codons encompassing modified adenosines are underlined and the corresponding amino acids (before/after editing) are indicated.
Figure Legend Snippet: A-to-I RNA editing at specific sites in the Rev and Tat coding sequence. The Rev and Tat coding sequence were analyzed by direct sequencing of RT–PCR products generated using as substrate the total RNA isolated as described in Figure 6 . Three independent co-transfection experiments with relative RT–PCR analysis were carried out. The edited adenosines found in the Rev and Tat coding sequence are numbered (relative to NL4-3) and shown in bold. The codons encompassing modified adenosines are underlined and the corresponding amino acids (before/after editing) are indicated.

Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Generated, Isolation, Cotransfection, Modification

A-to-I RNA editing at specific sites in the 5′ UTR of HIV-1 RNAs. The 5′ UTR sequence of HIV-1 was analyzed by direct sequencing of RT–PCR products generated using as substrate the total RNA isolated from 293T cells co-transfected with NL4-3 proviral DNA (15 μg) together with either pEGFP (8 μg) or pEGFP-ADAR1 (8 μg) or pEGFP-ADAR1 E/A (8 μg) plasmids. ( A ) Edited adenosines in the 5′ UTR sequence analyzed are shown in bold and underlined and their nucleotide position in the HIV-1 genome is indicated (NL4-3 numbering). ( B ) Representative DNA sequencing chromatograms of the RT–PCR products. Edited adenosines appear as mixture of A and G, and the estimated percentage of editing efficiency is indicated ( 30 ). Results shown are representative of three independent co-transfection experiments. ( C ) Schematic representation of the poly(A) hairpin (generated by using mfold server: http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi ) harbored in the 5′ UTR of HIV-1 RNA. Red arrows show adenosines that undergo ADAR1 editing and their relative nucleotide position in the HIV-1 genome.
Figure Legend Snippet: A-to-I RNA editing at specific sites in the 5′ UTR of HIV-1 RNAs. The 5′ UTR sequence of HIV-1 was analyzed by direct sequencing of RT–PCR products generated using as substrate the total RNA isolated from 293T cells co-transfected with NL4-3 proviral DNA (15 μg) together with either pEGFP (8 μg) or pEGFP-ADAR1 (8 μg) or pEGFP-ADAR1 E/A (8 μg) plasmids. ( A ) Edited adenosines in the 5′ UTR sequence analyzed are shown in bold and underlined and their nucleotide position in the HIV-1 genome is indicated (NL4-3 numbering). ( B ) Representative DNA sequencing chromatograms of the RT–PCR products. Edited adenosines appear as mixture of A and G, and the estimated percentage of editing efficiency is indicated ( 30 ). Results shown are representative of three independent co-transfection experiments. ( C ) Schematic representation of the poly(A) hairpin (generated by using mfold server: http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi ) harbored in the 5′ UTR of HIV-1 RNA. Red arrows show adenosines that undergo ADAR1 editing and their relative nucleotide position in the HIV-1 genome.

Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Generated, Isolation, Transfection, DNA Sequencing, Cotransfection

19) Product Images from "Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare"

Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00443

Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ( Holloway et al., 2011 ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.
Figure Legend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ( Holloway et al., 2011 ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.
Figure Legend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

Techniques Used: Polymerase Chain Reaction, Marker, Mouse Assay

20) Product Images from "Low Intracellular Iron Increases the Stability of Matriptase-2 *"

Article Title: Low Intracellular Iron Increases the Stability of Matriptase-2 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.611913

Iron deficiency does not alter the translation of Tmprss6 mRNA in the rat liver . A, acute and chronic iron deprivation does not alter Tmprss6 mRNA expression in rats. Weanling rats were fed either a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 3 days (day 3; n = 3) or 14 days (day 14; n = 5). Hepcidin ( Hamp ), Tmprss6 ( TM6 ), and mitochondrial aconitase ( m-acon ) mRNA expression in the liver were analyzed by qRT-PCR. Results are expressed as the amount of mRNA relative to β-actin in each sample. B, chronic iron deprivation increases MT2 protein in the liver. MT2 protein levels in the liver tissues from rats with chronic iron deprivation (day 14) as described above in A were detected by Western blot. β-Actin was included as a loading control. C–E, qRT-PCR analysis of m-Acon ( C ), Tmprss6 ( D ), and β-actin ( E ) mRNA in the fractions of polysome fractionation from the liver tissues of rats fed a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 3 days. The amount of mRNA in each fraction was expressed as the percentage of the combined total mRNA in all fractions. Each group consisted of three animals. F–H, qRT-PCR analysis of m-Acon ( F ), Tmprss6 ( G ), and β-actin ( H ) mRNA in the fractions of polysome fractionation from the liver tissues of rats fed a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 14 days. The amount of mRNA in each fraction was expressed as the percentage of the combined total mRNA in all fractions. Each group consisted of five animals. *, p
Figure Legend Snippet: Iron deficiency does not alter the translation of Tmprss6 mRNA in the rat liver . A, acute and chronic iron deprivation does not alter Tmprss6 mRNA expression in rats. Weanling rats were fed either a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 3 days (day 3; n = 3) or 14 days (day 14; n = 5). Hepcidin ( Hamp ), Tmprss6 ( TM6 ), and mitochondrial aconitase ( m-acon ) mRNA expression in the liver were analyzed by qRT-PCR. Results are expressed as the amount of mRNA relative to β-actin in each sample. B, chronic iron deprivation increases MT2 protein in the liver. MT2 protein levels in the liver tissues from rats with chronic iron deprivation (day 14) as described above in A were detected by Western blot. β-Actin was included as a loading control. C–E, qRT-PCR analysis of m-Acon ( C ), Tmprss6 ( D ), and β-actin ( E ) mRNA in the fractions of polysome fractionation from the liver tissues of rats fed a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 3 days. The amount of mRNA in each fraction was expressed as the percentage of the combined total mRNA in all fractions. Each group consisted of three animals. F–H, qRT-PCR analysis of m-Acon ( F ), Tmprss6 ( G ), and β-actin ( H ) mRNA in the fractions of polysome fractionation from the liver tissues of rats fed a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 14 days. The amount of mRNA in each fraction was expressed as the percentage of the combined total mRNA in all fractions. Each group consisted of five animals. *, p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Fractionation

Lack of change in Tmprss6 mRNA by BMP6, ID1, the BMP signaling, or iron. A, BMP6 did not induce TMPRSS6 mRNA expression in HepG2 cells. HepG2 cells were incubated in the presence of BMP6 at 0, 5, 25, and 50 ng/ml for 18 h, followed by qRT-PCR analysis of hepcidin, TMPRSS6, ID1 , and SMAD7 mRNA. The results are expressed as the amount of mRNA relative to β-actin in each sample. Results from four individual experiments are presented. B, increases in Bmp6 and Id1 expression did not induce Tmprss6 expression in mice. Eight-week-old Hjv- null male mice were injected with AAV8-Hjv vector containing a strong liver-specific promoter (−/− Hjv ) or the carrier vehicle (−/−). After 2 weeks, mice were euthanized for qRT-PCR analysis of Bmp6, Id1, Smad7, Tmprss6, and hepcidin mRNA in the liver. The results are expressed as the amount of mRNA relative to β-actin in each sample. Strain-, age-, and gender-matched wild-type ( wt ) mice were included as controls. Each group consisted of five animals. C and D, chronic iron load did not alter Tmprss6 expression in hepatocytes and the liver. Wild-type (9 weeks old) 129/S male mice were fed either a high iron rodent diet with 2% carbonyl iron (TD.08496; Harlan Laboratories) or a control iron rodent diet with 48 ppm iron (TD.80394; Harlan Laboratories) for 3 weeks. Tmprss6 mRNA in the whole liver and the isolated hepatocytes ( HC ), KC, SEC, and HSC ( C ), as well as hepcidin, Id1 , and Smad7 mRNA from the isolated hepatocytes of these mice ( D ) was analyzed by qRT-PCR. The results are expressed as the amount of mRNA relative to β-actin in each sample. Each group consisted of five animals. **, p
Figure Legend Snippet: Lack of change in Tmprss6 mRNA by BMP6, ID1, the BMP signaling, or iron. A, BMP6 did not induce TMPRSS6 mRNA expression in HepG2 cells. HepG2 cells were incubated in the presence of BMP6 at 0, 5, 25, and 50 ng/ml for 18 h, followed by qRT-PCR analysis of hepcidin, TMPRSS6, ID1 , and SMAD7 mRNA. The results are expressed as the amount of mRNA relative to β-actin in each sample. Results from four individual experiments are presented. B, increases in Bmp6 and Id1 expression did not induce Tmprss6 expression in mice. Eight-week-old Hjv- null male mice were injected with AAV8-Hjv vector containing a strong liver-specific promoter (−/− Hjv ) or the carrier vehicle (−/−). After 2 weeks, mice were euthanized for qRT-PCR analysis of Bmp6, Id1, Smad7, Tmprss6, and hepcidin mRNA in the liver. The results are expressed as the amount of mRNA relative to β-actin in each sample. Strain-, age-, and gender-matched wild-type ( wt ) mice were included as controls. Each group consisted of five animals. C and D, chronic iron load did not alter Tmprss6 expression in hepatocytes and the liver. Wild-type (9 weeks old) 129/S male mice were fed either a high iron rodent diet with 2% carbonyl iron (TD.08496; Harlan Laboratories) or a control iron rodent diet with 48 ppm iron (TD.80394; Harlan Laboratories) for 3 weeks. Tmprss6 mRNA in the whole liver and the isolated hepatocytes ( HC ), KC, SEC, and HSC ( C ), as well as hepcidin, Id1 , and Smad7 mRNA from the isolated hepatocytes of these mice ( D ) was analyzed by qRT-PCR. The results are expressed as the amount of mRNA relative to β-actin in each sample. Each group consisted of five animals. **, p

Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Mouse Assay, Injection, Plasmid Preparation, Isolation, Size-exclusion Chromatography

21) Product Images from "DNA methylation-mediated silencing of PU.1 in leukemia cells resistant to cell differentiation"

Article Title: DNA methylation-mediated silencing of PU.1 in leukemia cells resistant to cell differentiation

Journal: SpringerPlus

doi: 10.1186/2193-1801-2-392

Methylation status of the PU.1 promoter region. A) DNA chromatogram of the PU.1 upstream region using bisulfite-treated genomic DNA derived from MEL and MEL-R cell lines. Red circles highlight the cytosines of the four CpG islands of the MEL-R cell line that changed to thymines in the MEL-DS19 cell line after bisulfite treatment. B) Methylation analysis of the PCR products of the bisulfite-treated genomic DNA from A). The CG sequences were numbered according to (Shearstone et al. 2011 ) and their methylation status was determined.
Figure Legend Snippet: Methylation status of the PU.1 promoter region. A) DNA chromatogram of the PU.1 upstream region using bisulfite-treated genomic DNA derived from MEL and MEL-R cell lines. Red circles highlight the cytosines of the four CpG islands of the MEL-R cell line that changed to thymines in the MEL-DS19 cell line after bisulfite treatment. B) Methylation analysis of the PCR products of the bisulfite-treated genomic DNA from A). The CG sequences were numbered according to (Shearstone et al. 2011 ) and their methylation status was determined.

Techniques Used: Methylation, Derivative Assay, Polymerase Chain Reaction

DNA methylation status of the PU.1 locus regulatory region. A) A genomic map (not drawn to scale) depicts the regions containing the analyzed CpGs. The CpG colored in blue represent PCR amplified islands which however were not detected by the sequencing primers. CpG colored in red represent sequenced islands. B) Individual CpG methylation percentages of MEL DS19 and MEL-R cell lines.
Figure Legend Snippet: DNA methylation status of the PU.1 locus regulatory region. A) A genomic map (not drawn to scale) depicts the regions containing the analyzed CpGs. The CpG colored in blue represent PCR amplified islands which however were not detected by the sequencing primers. CpG colored in red represent sequenced islands. B) Individual CpG methylation percentages of MEL DS19 and MEL-R cell lines.

Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Amplification, Sequencing, CpG Methylation Assay

PCR probe to confirm the SFFV integration site within the PU.1 locus. A) Illustration of the multiple verification PCR amplifications using MEL genomic DNA as a template. Black arrows over the SFFV genome (gray box) indicate the pair of primers designed to amplify the SFFV-PU.1 junctions. Odd numbers 1 and 3 represent the primers used to identify the upstream integration junction and even numbers 2 and 4 represent the primers used for the downstream integration. Number 5 represents the long-range PCR (LR-PCR) used as a probe to confirm the complete SFFV integration. Black boxes correspond to the five exons of PU.1; the arrow above exon number one represents the initiation and direction of translation. B) Agarose gel electrophoresis performed using the primers schematized in A ) . C) Agarose gel electrophoresis of the LR-PCR probe to confirm SFFV integration; both the wild type (564 bp) and the integrated allele (6,859 bp) are visualized.
Figure Legend Snippet: PCR probe to confirm the SFFV integration site within the PU.1 locus. A) Illustration of the multiple verification PCR amplifications using MEL genomic DNA as a template. Black arrows over the SFFV genome (gray box) indicate the pair of primers designed to amplify the SFFV-PU.1 junctions. Odd numbers 1 and 3 represent the primers used to identify the upstream integration junction and even numbers 2 and 4 represent the primers used for the downstream integration. Number 5 represents the long-range PCR (LR-PCR) used as a probe to confirm the complete SFFV integration. Black boxes correspond to the five exons of PU.1; the arrow above exon number one represents the initiation and direction of translation. B) Agarose gel electrophoresis performed using the primers schematized in A ) . C) Agarose gel electrophoresis of the LR-PCR probe to confirm SFFV integration; both the wild type (564 bp) and the integrated allele (6,859 bp) are visualized.

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

22) Product Images from "Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8) *"

Article Title: Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.123158

Induction of Mct8 mRNA expression by retinoids in F9 cells. Results of quantitative RT-PCR for Mct8 and Gapdh mRNA are shown. A , time course of the induction of Mct8 mRNA by tRA. The inset graph shows induction within the first 24 h. Cells were treated
Figure Legend Snippet: Induction of Mct8 mRNA expression by retinoids in F9 cells. Results of quantitative RT-PCR for Mct8 and Gapdh mRNA are shown. A , time course of the induction of Mct8 mRNA by tRA. The inset graph shows induction within the first 24 h. Cells were treated

Techniques Used: Expressing, Quantitative RT-PCR

Effects of a transcription inhibitor, actinomycin D, on Mct8 mRNA expression in F9 cells. A , cells were treated with tRA (1 μ m ) and the indicated concentration of actinomycin D for 21 h, and quantitative RT-PCR of Mct8 was performed. Results were
Figure Legend Snippet: Effects of a transcription inhibitor, actinomycin D, on Mct8 mRNA expression in F9 cells. A , cells were treated with tRA (1 μ m ) and the indicated concentration of actinomycin D for 21 h, and quantitative RT-PCR of Mct8 was performed. Results were

Techniques Used: Expressing, Concentration Assay, Quantitative RT-PCR

TSS of Mct8 in F9 cells. A , results of 5′-RACE from tRA-treated F9 cells. Shown is agarose gel electrophoresis of the second round PCR with GeneRacer 5′ nested primer and 3′ +496 primer. Marker ( M ) was a 100-bp ladder. B , distribution
Figure Legend Snippet: TSS of Mct8 in F9 cells. A , results of 5′-RACE from tRA-treated F9 cells. Shown is agarose gel electrophoresis of the second round PCR with GeneRacer 5′ nested primer and 3′ +496 primer. Marker ( M ) was a 100-bp ladder. B , distribution

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

23) Product Images from "A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance"

Article Title: A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance

Journal: Human mutation

doi: 10.1002/humu.21071

PRPF31 mRNA isoforms in LCLs from members of family #1562 (A) A representative image of RT-PCR products, run on a 1.5% agarose gel, derived from the exon 13-exon 14 mRNA segment from 2 asymptomatic (III-9, III-11) and 1 affected (IV-16) individuals from family #1562, as well as from 1 CEPH individual (Ctrl). “M” is 100 bp ladder. Length and names of PCR products are indicated on the right side of the gel. (B) Schematic representation of the 3 mRNA isoforms from which PCR products are derived. Numbered rectangles and bold lines represent exons and introns of PRPF31 , respectively. Hatched rectangles designate parts of intron 13 that were retained (starts and ends are indicated by numbers). Numbering of stop codons for each mRNA isoform is based on the wild-type sequence. Depicted exons and introns are not to scale.
Figure Legend Snippet: PRPF31 mRNA isoforms in LCLs from members of family #1562 (A) A representative image of RT-PCR products, run on a 1.5% agarose gel, derived from the exon 13-exon 14 mRNA segment from 2 asymptomatic (III-9, III-11) and 1 affected (IV-16) individuals from family #1562, as well as from 1 CEPH individual (Ctrl). “M” is 100 bp ladder. Length and names of PCR products are indicated on the right side of the gel. (B) Schematic representation of the 3 mRNA isoforms from which PCR products are derived. Numbered rectangles and bold lines represent exons and introns of PRPF31 , respectively. Hatched rectangles designate parts of intron 13 that were retained (starts and ends are indicated by numbers). Numbering of stop codons for each mRNA isoform is based on the wild-type sequence. Depicted exons and introns are not to scale.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Derivative Assay, Polymerase Chain Reaction, Sequencing

24) Product Images from "A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance"

Article Title: A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance

Journal: Human mutation

doi: 10.1002/humu.21071

PRPF31 mRNA isoforms in LCLs from members of family #1562 (A) A representative image of RT-PCR products, run on a 1.5% agarose gel, derived from the exon 13-exon 14 mRNA segment from 2 asymptomatic (III-9, III-11) and 1 affected (IV-16) individuals from family #1562, as well as from 1 CEPH individual (Ctrl). “M” is 100 bp ladder. Length and names of PCR products are indicated on the right side of the gel. (B) Schematic representation of the 3 mRNA isoforms from which PCR products are derived. Numbered rectangles and bold lines represent exons and introns of PRPF31 , respectively. Hatched rectangles designate parts of intron 13 that were retained (starts and ends are indicated by numbers). Numbering of stop codons for each mRNA isoform is based on the wild-type sequence. Depicted exons and introns are not to scale.
Figure Legend Snippet: PRPF31 mRNA isoforms in LCLs from members of family #1562 (A) A representative image of RT-PCR products, run on a 1.5% agarose gel, derived from the exon 13-exon 14 mRNA segment from 2 asymptomatic (III-9, III-11) and 1 affected (IV-16) individuals from family #1562, as well as from 1 CEPH individual (Ctrl). “M” is 100 bp ladder. Length and names of PCR products are indicated on the right side of the gel. (B) Schematic representation of the 3 mRNA isoforms from which PCR products are derived. Numbered rectangles and bold lines represent exons and introns of PRPF31 , respectively. Hatched rectangles designate parts of intron 13 that were retained (starts and ends are indicated by numbers). Numbering of stop codons for each mRNA isoform is based on the wild-type sequence. Depicted exons and introns are not to scale.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Derivative Assay, Polymerase Chain Reaction, Sequencing

25) Product Images from "Transcriptional Regulation of PIK3CA Oncogene by NF-?B in Ovarian Cancer Microenvironment"

Article Title: Transcriptional Regulation of PIK3CA Oncogene by NF-?B in Ovarian Cancer Microenvironment

Journal: PLoS ONE

doi: 10.1371/journal.pone.0001758

TNF-α regulates PIK3CA expression via NF-κB pathway. A. H E staining of 2008 tumor reveals a prominent area of necrosis (N). B and C. Immunohistochemical staining of murine CD11b reveals macrophage infiltrate in a 2008 xenograft. CD11b+ cells infiltrate tumors in Ki67-negative regions in proximity of necrosis. C. High magnification from B. D. Double immunostaining of CD11b (green, FITC) and Ki67 (red, Texas Red) reveals CD11b+ macrophages mainly in non-proliferating Ki67-negative regions. E. ChIP analysis of NF-κB binding to the endogenous PIK3CA promoter. The arrows indicate the positions of the primers flanking −803 NF-κB binding site that were used in the ChIP assays. Cells were treated with TNF-α for 0, 30, or 90 min, and then chromatin protein-DNA complexes were cross-linked using formaldehyde. The purified nucleoprotein complexes were immunoprecipitated with p65 antibody or non-immune IgG and amplified by PCR. F. PIK3CA mRNA expression levels after stimulation with pro-inflammatory cytokine TNF-α. G. Illustration of the transcriptional regulation of PIK3CA by NF-κB.
Figure Legend Snippet: TNF-α regulates PIK3CA expression via NF-κB pathway. A. H E staining of 2008 tumor reveals a prominent area of necrosis (N). B and C. Immunohistochemical staining of murine CD11b reveals macrophage infiltrate in a 2008 xenograft. CD11b+ cells infiltrate tumors in Ki67-negative regions in proximity of necrosis. C. High magnification from B. D. Double immunostaining of CD11b (green, FITC) and Ki67 (red, Texas Red) reveals CD11b+ macrophages mainly in non-proliferating Ki67-negative regions. E. ChIP analysis of NF-κB binding to the endogenous PIK3CA promoter. The arrows indicate the positions of the primers flanking −803 NF-κB binding site that were used in the ChIP assays. Cells were treated with TNF-α for 0, 30, or 90 min, and then chromatin protein-DNA complexes were cross-linked using formaldehyde. The purified nucleoprotein complexes were immunoprecipitated with p65 antibody or non-immune IgG and amplified by PCR. F. PIK3CA mRNA expression levels after stimulation with pro-inflammatory cytokine TNF-α. G. Illustration of the transcriptional regulation of PIK3CA by NF-κB.

Techniques Used: Expressing, Staining, Immunohistochemistry, Double Immunostaining, Chromatin Immunoprecipitation, Binding Assay, Purification, Immunoprecipitation, Amplification, Polymerase Chain Reaction

Identification and characterization of the human PIK3CA promoters. A. Illustration of the structure of human PIK3CA gene and its 5′ upstream regulatory region. B. A region highly rich in GC is found in the PIK3CA 5′TRR. C. Illustration of the primers used for mapping RT-PCR of the PIK3CA transcriptional start site (SST). D. Results of mapping RT-PCR. There is no band between the forward primer F1 located upstream of the SST and the reverse primer R located on exon 1 of PIK3CA . The right size bands could be detected between primer F2 or F3 (both located downstream of SST) and reverse primer R. E. A small splicing variant is found in the 5′UTR of human PIK3CA gene, which can also be detected by mapping RT-PCR (primers F2 and R). F. Summarized results of the transcriptional activity of PIK3CA TRR fragments.
Figure Legend Snippet: Identification and characterization of the human PIK3CA promoters. A. Illustration of the structure of human PIK3CA gene and its 5′ upstream regulatory region. B. A region highly rich in GC is found in the PIK3CA 5′TRR. C. Illustration of the primers used for mapping RT-PCR of the PIK3CA transcriptional start site (SST). D. Results of mapping RT-PCR. There is no band between the forward primer F1 located upstream of the SST and the reverse primer R located on exon 1 of PIK3CA . The right size bands could be detected between primer F2 or F3 (both located downstream of SST) and reverse primer R. E. A small splicing variant is found in the 5′UTR of human PIK3CA gene, which can also be detected by mapping RT-PCR (primers F2 and R). F. Summarized results of the transcriptional activity of PIK3CA TRR fragments.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Variant Assay, Activity Assay

26) Product Images from "Disruption of the carA gene in Pseudomonas syringae results in reduced fitness and alters motility"

Article Title: Disruption of the carA gene in Pseudomonas syringae results in reduced fitness and alters motility

Journal: BMC Microbiology

doi: 10.1186/s12866-016-0819-z

Expression of P32 and carA in wild-type DC3000 compared to ΔargR mutant using qRT-PCR. The dark gray bars represent the ratios of the transcripts comparing ΔargR mutant to the WT at mid-log phase, and the light gray bars represent the ratios of the transcripts comparing Δ argR mutant to the WT at stationary phase. RNA samples were normalized using gap1 . The Δ argR mutant shows increased levels of P32 and carA transcript compared to the WT at mid-log phase. The levels of P32 and carA transcripts were analyzed by calculating the fold difference of transcript levels between WT and Δ argR mutant using the Δ C t method. Data shown are the average and standard deviation of three independent biological replicates
Figure Legend Snippet: Expression of P32 and carA in wild-type DC3000 compared to ΔargR mutant using qRT-PCR. The dark gray bars represent the ratios of the transcripts comparing ΔargR mutant to the WT at mid-log phase, and the light gray bars represent the ratios of the transcripts comparing Δ argR mutant to the WT at stationary phase. RNA samples were normalized using gap1 . The Δ argR mutant shows increased levels of P32 and carA transcript compared to the WT at mid-log phase. The levels of P32 and carA transcripts were analyzed by calculating the fold difference of transcript levels between WT and Δ argR mutant using the Δ C t method. Data shown are the average and standard deviation of three independent biological replicates

Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Standard Deviation

Co-transcription of P32 and carA . a Map of the genomic region containing dapB , P32 and carA in DC3000. The locations and orientations of RT- and PCR primers are indicated. b Agarose gel electrophoresis result of the RT-PCR experiments using the primers pairs indicated. The expected length of the PCR products for the primer pairs are as follows: Primer pair 1 and 2, ~54 bps; primer pair 1 and 4, ~370 bps; and primer pair 3 and 4, ~209 bps. Control reactions in which reverse transcriptase was omitted were performed for each primer set and RNA sample
Figure Legend Snippet: Co-transcription of P32 and carA . a Map of the genomic region containing dapB , P32 and carA in DC3000. The locations and orientations of RT- and PCR primers are indicated. b Agarose gel electrophoresis result of the RT-PCR experiments using the primers pairs indicated. The expected length of the PCR products for the primer pairs are as follows: Primer pair 1 and 2, ~54 bps; primer pair 1 and 4, ~370 bps; and primer pair 3 and 4, ~209 bps. Control reactions in which reverse transcriptase was omitted were performed for each primer set and RNA sample

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

27) Product Images from "Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells *"

Article Title: Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.050757

Expression of murine Tlr11 gene in epithelial cells and determination of the Tlr11 transcription start site by 5′-RACE PCR. A, expression of Tlr11 in murine epithelial cells. Total RNA from mouse embryo fibroblast ( MEF ), macrophage Raw 264.7,
Figure Legend Snippet: Expression of murine Tlr11 gene in epithelial cells and determination of the Tlr11 transcription start site by 5′-RACE PCR. A, expression of Tlr11 in murine epithelial cells. Total RNA from mouse embryo fibroblast ( MEF ), macrophage Raw 264.7,

Techniques Used: Expressing, Polymerase Chain Reaction

Effects of the knocked down expression of ESE-1, ESE-3, and IRF8 on Tlr11 endogenous expression and promoter activity. A, real time RT-PCR analysis of Tlr11 expression in knocked down cells. Total RNA extracted from ESE-1, ESE-3, and IRF8 knocked down
Figure Legend Snippet: Effects of the knocked down expression of ESE-1, ESE-3, and IRF8 on Tlr11 endogenous expression and promoter activity. A, real time RT-PCR analysis of Tlr11 expression in knocked down cells. Total RNA extracted from ESE-1, ESE-3, and IRF8 knocked down

Techniques Used: Expressing, Activity Assay, Quantitative RT-PCR

28) Product Images from "A missense mutation (Q279R) in the Fumarylacetoacetate Hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation"

Article Title: A missense mutation (Q279R) in the Fumarylacetoacetate Hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation

Journal: BMC Genetics

doi: 10.1186/1471-2156-2-9

Mutation analysis in different liver regions. DNA was extracted from different liver regions and amplified by PCR. PCR products were digested with either Alu I to detect IVS6-1g- > t or with Msp I to detect Q279R. For IVS6-1 g- > t, the same heterozygous pattern is seen in both the reverted nodule (NT), tumor section (T) and fibroblast DNA (F), showing 3 bands, one at 156-, another at 104- and the last at 75-bp. The control (wt/wt) shows two bands, one at 156- and the other at 75-bp, indicating the absence of IVS6-1g- > t (M: molecular weight marker, 100- and 200-bp). For Q279R both the 78- and 58-bp bands are seen in the tumor section (T) and fibroblast DNA (F) indicating an heterozygous genotype while only the 78-bp wild-type band is seen in the control (wt/wt). In the region suspected of reversion (NT), a strong 78-bp wild-type band is seen with a weak 58-bp mutated band (M: molecular weight marker, 100-bp).
Figure Legend Snippet: Mutation analysis in different liver regions. DNA was extracted from different liver regions and amplified by PCR. PCR products were digested with either Alu I to detect IVS6-1g- > t or with Msp I to detect Q279R. For IVS6-1 g- > t, the same heterozygous pattern is seen in both the reverted nodule (NT), tumor section (T) and fibroblast DNA (F), showing 3 bands, one at 156-, another at 104- and the last at 75-bp. The control (wt/wt) shows two bands, one at 156- and the other at 75-bp, indicating the absence of IVS6-1g- > t (M: molecular weight marker, 100- and 200-bp). For Q279R both the 78- and 58-bp bands are seen in the tumor section (T) and fibroblast DNA (F) indicating an heterozygous genotype while only the 78-bp wild-type band is seen in the control (wt/wt). In the region suspected of reversion (NT), a strong 78-bp wild-type band is seen with a weak 58-bp mutated band (M: molecular weight marker, 100-bp).

Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, Molecular Weight, Marker

Analysis of the splicing pattern obtained with the minigenes. A- The splicing K7 consists of exon 1 of β-globin and its downstream intronic sequences joined to β-globin exon 3 and its upstream intronic sequences. Exon 9, with or without the Q279R mutation was inserted in K7 at the intronic junction. HeLa cells were transiently transfected with both constructs, the wild-type Q279Q-K7 and Q279R-containing Q279R-K7. After 24 hours, cells were harvested and the splicing pattern of each minigene was examined by RT-PCR analysis of the transcripts. Exons are represented by boxes and introns by lines. The primers used for RT-PCR are indicated at each end of the splicing K7. B- Total RNA extracted from transfected HeLa cells was amplified with HG1S and HG3AS. Plasmidic DNA Q279Q-K7 and Q279R-K7 (pDNA) were also amplified as a control. The band obtained for Q279Q-K7 transfected cells (RT+) is of expected size, in contrast to the band obtained in the case of Q279R-K7 (RT+) transfected cells, which is of lower molecular weight. RT - serves as a negative control: the reverse transcription reaction was performed without any enzyme. In the two RT - fractions, the amplification of about 900-bp is due to plasmidic DNA contamination.
Figure Legend Snippet: Analysis of the splicing pattern obtained with the minigenes. A- The splicing K7 consists of exon 1 of β-globin and its downstream intronic sequences joined to β-globin exon 3 and its upstream intronic sequences. Exon 9, with or without the Q279R mutation was inserted in K7 at the intronic junction. HeLa cells were transiently transfected with both constructs, the wild-type Q279Q-K7 and Q279R-containing Q279R-K7. After 24 hours, cells were harvested and the splicing pattern of each minigene was examined by RT-PCR analysis of the transcripts. Exons are represented by boxes and introns by lines. The primers used for RT-PCR are indicated at each end of the splicing K7. B- Total RNA extracted from transfected HeLa cells was amplified with HG1S and HG3AS. Plasmidic DNA Q279Q-K7 and Q279R-K7 (pDNA) were also amplified as a control. The band obtained for Q279Q-K7 transfected cells (RT+) is of expected size, in contrast to the band obtained in the case of Q279R-K7 (RT+) transfected cells, which is of lower molecular weight. RT - serves as a negative control: the reverse transcription reaction was performed without any enzyme. In the two RT - fractions, the amplification of about 900-bp is due to plasmidic DNA contamination.

Techniques Used: Mutagenesis, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Amplification, Molecular Weight, Negative Control

29) Product Images from "L1 retrotransposon-mediated stable gene silencing"

Article Title: L1 retrotransposon-mediated stable gene silencing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gni056

Inhibition of exogenous GFP expression using L1-based RNAi system. ( A ) Real-time RT–PCR results of GFP mRNA expression in stable HeLa lines expressing control or GFP-targeted siRNA ( n = 6). ( B ) Western-blot analysis of GFP expression in control or knockdown clones. ( C ) Typical FACS dot plots of GFP expression in control and knockdown clones. ( D ) Normalized GFP fluorescence in control and knockdown clones by FACS ( n = 4).
Figure Legend Snippet: Inhibition of exogenous GFP expression using L1-based RNAi system. ( A ) Real-time RT–PCR results of GFP mRNA expression in stable HeLa lines expressing control or GFP-targeted siRNA ( n = 6). ( B ) Western-blot analysis of GFP expression in control or knockdown clones. ( C ) Typical FACS dot plots of GFP expression in control and knockdown clones. ( D ) Normalized GFP fluorescence in control and knockdown clones by FACS ( n = 4).

Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Clone Assay, FACS, Fluorescence

Inhibition of endogenous GAPDH expression using L1-based RNAi system. ( A ) Real-time RT–PCR results of GAPDH mRNA expression in stable HeLa lines expressing control, GFP-targeted or GAPDH-targeted siRNA ( n = 6). ( B ) Western-blot analysis of GAPDH protein expression in control or GAPDH knockdown clones. ( C ) Immunofluorescent staining of GAPDH (green) and cytokeratin (red) in control and knockdown cells. Nucleus is counterstained with DAPI (blue).
Figure Legend Snippet: Inhibition of endogenous GAPDH expression using L1-based RNAi system. ( A ) Real-time RT–PCR results of GAPDH mRNA expression in stable HeLa lines expressing control, GFP-targeted or GAPDH-targeted siRNA ( n = 6). ( B ) Western-blot analysis of GAPDH protein expression in control or GAPDH knockdown clones. ( C ) Immunofluorescent staining of GAPDH (green) and cytokeratin (red) in control and knockdown cells. Nucleus is counterstained with DAPI (blue).

Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Clone Assay, Staining

30) Product Images from "The involvement of replication in single stranded oligonucleotide-mediated gene repair"

Article Title: The involvement of replication in single stranded oligonucleotide-mediated gene repair

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl852

Chain-terminating ddC residue prevents replicative extension in vitro . Primers containing 6 phosphothioate linkages at each terminus (PT SSO) or 6 phosphothioate linkages at each terminus and a 3′-dideoxycytidine residue (PT+ddC SSO) were used to amplify a 196 bp fragment using pGKfrtmCM(−) as template and mCM(+)DT2 as the reverse primer, in a standard PCR reaction (see Table 6 in Supplementary material for primer sequences). PCRs were performed with the modified SSOs present at three different concentrations (1, 10 or 100 ng per reaction). Results show that the chain-terminating ddC nucleotide on the PT+ddC SSO is sufficient to prevent replicative extension by a DNA polymerase endowed with proofreading activity.
Figure Legend Snippet: Chain-terminating ddC residue prevents replicative extension in vitro . Primers containing 6 phosphothioate linkages at each terminus (PT SSO) or 6 phosphothioate linkages at each terminus and a 3′-dideoxycytidine residue (PT+ddC SSO) were used to amplify a 196 bp fragment using pGKfrtmCM(−) as template and mCM(+)DT2 as the reverse primer, in a standard PCR reaction (see Table 6 in Supplementary material for primer sequences). PCRs were performed with the modified SSOs present at three different concentrations (1, 10 or 100 ng per reaction). Results show that the chain-terminating ddC nucleotide on the PT+ddC SSO is sufficient to prevent replicative extension by a DNA polymerase endowed with proofreading activity.

Techniques Used: In Vitro, Polymerase Chain Reaction, Modification, Activity Assay

Verification of SSO incorporation into its homologous DNA target ( A ) A schematic illustration of the experimental procedure. Biotinylated recombination products were purified using magnetic streptavidin beads. The presence of (corrected) pmKan was confirmed by the detection of a 496 bp PCR product. ( B ) pmKan and ddH 2 O were used as templates for the negative and positive PCR controls (lanes 2 and 3 respectively). DY380/pmKan cells were incubated at 42°C for 15 min to induce λ-Red protein expression prior to electroporation with biotinylated-SSO (lane 6) or unmodified SSO (lane 4). As a control, DY380/pmKan cells that had been incubated at 32°C for 15 min (i.e. no λ-Red induction) were similarly electroporated with biotinylated-SSO (lane 5). Plasmid DNA were extracted from the electroporated cells after a 15 min recovery period. Three independent experiments were performed; a representative experiment is shown.
Figure Legend Snippet: Verification of SSO incorporation into its homologous DNA target ( A ) A schematic illustration of the experimental procedure. Biotinylated recombination products were purified using magnetic streptavidin beads. The presence of (corrected) pmKan was confirmed by the detection of a 496 bp PCR product. ( B ) pmKan and ddH 2 O were used as templates for the negative and positive PCR controls (lanes 2 and 3 respectively). DY380/pmKan cells were incubated at 42°C for 15 min to induce λ-Red protein expression prior to electroporation with biotinylated-SSO (lane 6) or unmodified SSO (lane 4). As a control, DY380/pmKan cells that had been incubated at 32°C for 15 min (i.e. no λ-Red induction) were similarly electroporated with biotinylated-SSO (lane 5). Plasmid DNA were extracted from the electroporated cells after a 15 min recovery period. Three independent experiments were performed; a representative experiment is shown.

Techniques Used: Purification, Polymerase Chain Reaction, Incubation, Expressing, Electroporation, Plasmid Preparation

31) Product Images from "Physical and Functional Interactions between the Histone H3K4 Demethylase KDM5A and the Nucleosome Remodeling and Deacetylase (NuRD) Complex"

Article Title: Physical and Functional Interactions between the Histone H3K4 Demethylase KDM5A and the Nucleosome Remodeling and Deacetylase (NuRD) Complex

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.573725

Comparison of genes regulated by KDM5A, SIN3B, or CHD4. A and B, expression levels of three KDM5 family genes in HeLa, HEK293T, U2OS, or MCF7 cells. The levels were determined by quantitative real time-PCR (qRT-PCR) ( A ) and immunoblotting using the indicated antibodies ( B ). The RT-PCR values were normalized to the GAPDH level. Error bars represent standard deviations of three biological replicates. C, HeLa cells were treated with the control ( siCont ) or indicated siRNAs, and the levels of endogenous KDM5A, CHD4, SIN3B, or tubulin (loading control) were analyzed by Western blotting. D, Venn diagram representing the genes dysregulated by KDM5A-, CHD4-, or SIN3B-knockdown in HeLa cells. Genes displaying ≥1.5-fold changed expression and a p value
Figure Legend Snippet: Comparison of genes regulated by KDM5A, SIN3B, or CHD4. A and B, expression levels of three KDM5 family genes in HeLa, HEK293T, U2OS, or MCF7 cells. The levels were determined by quantitative real time-PCR (qRT-PCR) ( A ) and immunoblotting using the indicated antibodies ( B ). The RT-PCR values were normalized to the GAPDH level. Error bars represent standard deviations of three biological replicates. C, HeLa cells were treated with the control ( siCont ) or indicated siRNAs, and the levels of endogenous KDM5A, CHD4, SIN3B, or tubulin (loading control) were analyzed by Western blotting. D, Venn diagram representing the genes dysregulated by KDM5A-, CHD4-, or SIN3B-knockdown in HeLa cells. Genes displaying ≥1.5-fold changed expression and a p value

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot

Validation of genes regulated by KDM5A and NuRD. A, categorization of the genes that were dysregulated in both siKDM5A- and siCHD4-treated cells. The genes were classified into four categories according to the effect of each siRNA treatment. B–E, expression levels of representative genes of each category in siRNA-treated HeLa ( B ), U2OS ( C ), HEK293T ( D ), and MCF7 ( E ) cells. The levels were determined by qRT-PCR after each knockdown and were compared with the levels of control siRNA-treated cells. The values were normalized to GAPDH levels. Error bars represent standard deviations of three biological replicates.
Figure Legend Snippet: Validation of genes regulated by KDM5A and NuRD. A, categorization of the genes that were dysregulated in both siKDM5A- and siCHD4-treated cells. The genes were classified into four categories according to the effect of each siRNA treatment. B–E, expression levels of representative genes of each category in siRNA-treated HeLa ( B ), U2OS ( C ), HEK293T ( D ), and MCF7 ( E ) cells. The levels were determined by qRT-PCR after each knockdown and were compared with the levels of control siRNA-treated cells. The values were normalized to GAPDH levels. Error bars represent standard deviations of three biological replicates.

Techniques Used: Expressing, Quantitative RT-PCR

32) Product Images from "Superinfection Exclusion in Duck Hepatitis B Virus Infection Is Mediated by the Large Surface Antigen"

Article Title: Superinfection Exclusion in Duck Hepatitis B Virus Infection Is Mediated by the Large Surface Antigen

Journal: Journal of Virology

doi: 10.1128/JVI.78.15.7925-7937.2004

Single-cell PCR analysis of the viral population within DHBV-infected hepatocytes. (A) PCR analysis of individual hepatocytes obtained from a duck coinfected with DHBV and DHBV-ClaI. PCR products were separated by size on 1.2% agarose gels, transferred
Figure Legend Snippet: Single-cell PCR analysis of the viral population within DHBV-infected hepatocytes. (A) PCR analysis of individual hepatocytes obtained from a duck coinfected with DHBV and DHBV-ClaI. PCR products were separated by size on 1.2% agarose gels, transferred

Techniques Used: Polymerase Chain Reaction, Infection

Infection of uninfected and congenitally infected ducklings with DHBV-ClaI. Ducklings were inoculated i.m. with 2 × 10 10 VGE of either DHBV-ClaI alone (A) or a mixture of DHBV-ClaI and DHBV (B). Serum viral DNA was extracted and amplified by PCR.
Figure Legend Snippet: Infection of uninfected and congenitally infected ducklings with DHBV-ClaI. Ducklings were inoculated i.m. with 2 × 10 10 VGE of either DHBV-ClaI alone (A) or a mixture of DHBV-ClaI and DHBV (B). Serum viral DNA was extracted and amplified by PCR.

Techniques Used: Infection, Amplification, Polymerase Chain Reaction

33) Product Images from "Recurrent (2;2) and (2;8) Translocations in Rhabdomyosarcoma without the Canonical PAX-FOXO1 fuse PAX3 to Members of the Nuclear Receptor Transcriptional Coactivator (NCOA) Family"

Article Title: Recurrent (2;2) and (2;8) Translocations in Rhabdomyosarcoma without the Canonical PAX-FOXO1 fuse PAX3 to Members of the Nuclear Receptor Transcriptional Coactivator (NCOA) Family

Journal: Genes, chromosomes & cancer

doi: 10.1002/gcc.20731

Representative FISH analysis of t(2;2)(p23;q35) and t(2;8)(q35;q12) translocations. A. FISH analysis of Case 1 with the custom designed NCOA1 break-apart probe set demonstrates split orange and green signals (arrows) indicative of a rearrangement of this locus. B. FISH analysis of Case 1 with the PAX3 spanning probe set in orange and the NCOA1 spanning probe set in green demonstrates the presence of juxtaposed or fused orange and green signals consistent with the RT-PCR findings of a PAX3-NCOA1 fusion transcript in this case. C and D. FISH analyses of Case 4 with the custom designed PAX3 and NCOA2 break-apart probe sets, respectively, demonstrate split orange and green signals (arrows) indicative of a rearrangement of each of these loci.
Figure Legend Snippet: Representative FISH analysis of t(2;2)(p23;q35) and t(2;8)(q35;q12) translocations. A. FISH analysis of Case 1 with the custom designed NCOA1 break-apart probe set demonstrates split orange and green signals (arrows) indicative of a rearrangement of this locus. B. FISH analysis of Case 1 with the PAX3 spanning probe set in orange and the NCOA1 spanning probe set in green demonstrates the presence of juxtaposed or fused orange and green signals consistent with the RT-PCR findings of a PAX3-NCOA1 fusion transcript in this case. C and D. FISH analyses of Case 4 with the custom designed PAX3 and NCOA2 break-apart probe sets, respectively, demonstrate split orange and green signals (arrows) indicative of a rearrangement of each of these loci.

Techniques Used: Fluorescence In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction

Representative RT-PCR and sequence analyses for chimeric transcripts in Cases 1-5. A. Detection of PAX3-NCOA1 transcripts in Case 1 (lanes 1 and 2); 2 (lanes 3 and 4) and 3 (lanes 5 and 6). Primers used for lanes 1, 3 and 5 were PAX3-32 and NCOA1-33; for lanes 2, 4 and 6, PAX3-34 and NCOA1-35. Detection of PAX3-NCOA2 transcripts in Cases 4 (lane 7) and 5 (lane 8). Primers used were PAX3-41 and NCOA2-48. B . Sequence alignment of the PAX3-NCOA1 and PAX3-NCOA2 breakpoint regions. Arrows depict the fusion point. Single letter amino acid code is displayed beneath the nucleotide sequence.
Figure Legend Snippet: Representative RT-PCR and sequence analyses for chimeric transcripts in Cases 1-5. A. Detection of PAX3-NCOA1 transcripts in Case 1 (lanes 1 and 2); 2 (lanes 3 and 4) and 3 (lanes 5 and 6). Primers used for lanes 1, 3 and 5 were PAX3-32 and NCOA1-33; for lanes 2, 4 and 6, PAX3-34 and NCOA1-35. Detection of PAX3-NCOA2 transcripts in Cases 4 (lane 7) and 5 (lane 8). Primers used were PAX3-41 and NCOA2-48. B . Sequence alignment of the PAX3-NCOA1 and PAX3-NCOA2 breakpoint regions. Arrows depict the fusion point. Single letter amino acid code is displayed beneath the nucleotide sequence.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing

34) Product Images from "The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms"

Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002534

PCR analysis of integration junctions from lariat and linear intron RNA retrohoming in wild-type and mutant strains. Retrohoming assays with lariat and linear RNPs were done as described in Figure 2A and Materials and Methods , and DNA was extracted from 80 pooled embryos for each strain. 5′- and 3′-integration junctions were amplified by PCR, using primers that flank the junction (5′junction, forward primer P1 and reverse primer LtrB933a; 3′ junction, forward primer P3 and reverse primer P4; see Materials and Methods ). The PCR products were analyzed in a 1% agarose gel, which was stained with ethidium bromide. Precise 5′ and 3′ junctions for lariat intron RNA retrohoming and precise 3′ junctions for linear intron RNA retrohoming were confirmed by sequencing junctions from at least 10 randomly selected Tet R +Amp R colonies or PCR products from pooled embryos for all strains (not shown).
Figure Legend Snippet: PCR analysis of integration junctions from lariat and linear intron RNA retrohoming in wild-type and mutant strains. Retrohoming assays with lariat and linear RNPs were done as described in Figure 2A and Materials and Methods , and DNA was extracted from 80 pooled embryos for each strain. 5′- and 3′-integration junctions were amplified by PCR, using primers that flank the junction (5′junction, forward primer P1 and reverse primer LtrB933a; 3′ junction, forward primer P3 and reverse primer P4; see Materials and Methods ). The PCR products were analyzed in a 1% agarose gel, which was stained with ethidium bromide. Precise 5′ and 3′ junctions for lariat intron RNA retrohoming and precise 3′ junctions for linear intron RNA retrohoming were confirmed by sequencing junctions from at least 10 randomly selected Tet R +Amp R colonies or PCR products from pooled embryos for all strains (not shown).

Techniques Used: Polymerase Chain Reaction, Mutagenesis, Amplification, Agarose Gel Electrophoresis, Staining, Sequencing

Sequences of 5′-integration junctions from linear intron RNA retrohoming in wild-type and mutant strains. 5′-integration junctions of DNA extracted from 80 pooled embryos for each strain were amplified by PCR, as described in Figure 3 , then TOPA-TA cloned, amplified by colony PCR, and sequenced, as described in Materials and Methods . (A) wild-type w 1118 ; (B) lig4 − ; (C) ku70 − ; (D) lig4 − ; P{lig4 + }; (E) wild-type Or-R; (F) polQ − . Inserted or mutant nucleotide residues are shown in lower case letters; microhomologies between intron and exon end sequences prior to ligation are shown in parentheses; and inserted sequences that match or are complementary to nearby 5′-exon or intron sequences are underlined. Freq., frequency of occurrence.
Figure Legend Snippet: Sequences of 5′-integration junctions from linear intron RNA retrohoming in wild-type and mutant strains. 5′-integration junctions of DNA extracted from 80 pooled embryos for each strain were amplified by PCR, as described in Figure 3 , then TOPA-TA cloned, amplified by colony PCR, and sequenced, as described in Materials and Methods . (A) wild-type w 1118 ; (B) lig4 − ; (C) ku70 − ; (D) lig4 − ; P{lig4 + }; (E) wild-type Or-R; (F) polQ − . Inserted or mutant nucleotide residues are shown in lower case letters; microhomologies between intron and exon end sequences prior to ligation are shown in parentheses; and inserted sequences that match or are complementary to nearby 5′-exon or intron sequences are underlined. Freq., frequency of occurrence.

Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, Clone Assay, Ligation

35) Product Images from "The Jacob2 Lectin of the Entamoeba histolytica Cyst Wall Binds Chitin and Is Polymorphic"

Article Title: The Jacob2 Lectin of the Entamoeba histolytica Cyst Wall Binds Chitin and Is Polymorphic

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0000750

Ser-rich domains of EhJacob2 are polymorphic. Amplification products were generated using PCR primers flanking the Ser-rich region between the second and third chitin-binding domains of Jacob2. A. Jacob2 PCR products from axenized Eh isolates (HM-1:IMSS, HK-9, 200:NIH, and SD157) and Ed isolate (SAW760) have distinct mobilities on agarose gels. B. Jacob2 PCR products from clinical Eh isolates also have distinct mobilities.
Figure Legend Snippet: Ser-rich domains of EhJacob2 are polymorphic. Amplification products were generated using PCR primers flanking the Ser-rich region between the second and third chitin-binding domains of Jacob2. A. Jacob2 PCR products from axenized Eh isolates (HM-1:IMSS, HK-9, 200:NIH, and SD157) and Ed isolate (SAW760) have distinct mobilities on agarose gels. B. Jacob2 PCR products from clinical Eh isolates also have distinct mobilities.

Techniques Used: Amplification, Generated, Polymerase Chain Reaction, Binding Assay

EhJacob2 PCR products are distinct for each axenized strain. A. Coded representations of EhJacob2 repeats from PCR products shown in Fig. 3 . Complete sequences were obtained for HM-1:IMSS and SD157. Gaps in the middle of sequences in the HK-9 and 200:NIH products are marked. B. Five EhJacob2 repeats are each assigned a letter (A to E) and a color (as described in Fig. 1 ). The nucleotide sequences coding for each repeat are numbered in the order of their frequency of occurrence in the sequenced products.
Figure Legend Snippet: EhJacob2 PCR products are distinct for each axenized strain. A. Coded representations of EhJacob2 repeats from PCR products shown in Fig. 3 . Complete sequences were obtained for HM-1:IMSS and SD157. Gaps in the middle of sequences in the HK-9 and 200:NIH products are marked. B. Five EhJacob2 repeats are each assigned a letter (A to E) and a color (as described in Fig. 1 ). The nucleotide sequences coding for each repeat are numbered in the order of their frequency of occurrence in the sequenced products.

Techniques Used: Polymerase Chain Reaction

36) Product Images from "Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs"

Article Title: Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.211399398

PCR analysis of the genomic DNA extracted from HMEC-1 cells nontransduced or transduced with various retroviral vectors. Different combinations of primers were used to amplify HHO-1, neo r , or chimeric vector/HHO-1 DNA fragments. P1 and P2, primers for amplifying LXSN vector sequence; P3 and P4, primers for neo r gene; P5 and P6, primers for HHO-1 DNA fragment. The combination of P1 and P5 for HHO-1-AS-transduced HMEC-1 cells (HMEC/HOP-HHO-1-AS) detected a 2,441-bp signal that contains partial vector sequence, HOP, and a partial HHO-1-AS DNA fragment. Similarly, a 2,260-bp PCR product containing partial vector sequence, HOP, and a partial HHO-1 gene fragment was amplified from HHO-1-transduced cells (HMEC/HOP-HHO-1) when primers P1 and P6 were used. The primers P1 and P2 amplified a 1,661-bp PCR product from control vector (LSN-HOP)-transduced HMEC-1 cells (HMEC/HOP). The 313-bp neo r DNA fragments were detected in all three kinds of cells mentioned above by using primers P3 and P4.
Figure Legend Snippet: PCR analysis of the genomic DNA extracted from HMEC-1 cells nontransduced or transduced with various retroviral vectors. Different combinations of primers were used to amplify HHO-1, neo r , or chimeric vector/HHO-1 DNA fragments. P1 and P2, primers for amplifying LXSN vector sequence; P3 and P4, primers for neo r gene; P5 and P6, primers for HHO-1 DNA fragment. The combination of P1 and P5 for HHO-1-AS-transduced HMEC-1 cells (HMEC/HOP-HHO-1-AS) detected a 2,441-bp signal that contains partial vector sequence, HOP, and a partial HHO-1-AS DNA fragment. Similarly, a 2,260-bp PCR product containing partial vector sequence, HOP, and a partial HHO-1 gene fragment was amplified from HHO-1-transduced cells (HMEC/HOP-HHO-1) when primers P1 and P6 were used. The primers P1 and P2 amplified a 1,661-bp PCR product from control vector (LSN-HOP)-transduced HMEC-1 cells (HMEC/HOP). The 313-bp neo r DNA fragments were detected in all three kinds of cells mentioned above by using primers P3 and P4.

Techniques Used: Polymerase Chain Reaction, Transduction, Plasmid Preparation, Sequencing, Amplification

( A ) Northern blot analysis of RNA obtained from PA317 and NIH 3T3 cells nontransduced or transduced with retroviral vector LSN-HHO-1 or LXSN. Lanes 1 and 2, PA317/HHO-1 cells; lane 3, control PA317 retroviral packaging cells; lane 4, PA317/LXSN cells; lane 5, NIH 3T3/LXSN cells; lanes 6 and 7, NIH 3T3/HHO-1 cells; lane 8, control NIH 3T3 cells. ( B ) Reverse transcription (RT)-PCR analysis of PT67 retroviral packaging cells transduced with different retroviral vectors. Lanes 1 and 6, PT67 cells; lanes 2 and 7, PT67/LXSN cells; lanes 3 and 8, PT67/LSN-HOP cells; lanes 4 and 9, PT67/LSN-HOP-HHO-1 cells; Lanes 5 and 10, PT67/LSN-HOP-HHO-1-AS cells. m, Hin dIII-digested λ DNA marker; M, 100-bp DNA ruler; neo r , RT-PCR products of neomycin-resistance gene; HO-1, human HO-1 RT-PCR products. ( C ) Detection of human (h)HO-1 and G3PDH transcripts by Northern blot analysis in RLMV cells transduced with retroviral vectors LSN-HHO-1 or LXSN. Lanes 1, 2, and 5, RLMV cells transduced with LSN-HHO-1; lane 3, control RLMV cells; lane 4, RLMV cells transduced with LXSN.
Figure Legend Snippet: ( A ) Northern blot analysis of RNA obtained from PA317 and NIH 3T3 cells nontransduced or transduced with retroviral vector LSN-HHO-1 or LXSN. Lanes 1 and 2, PA317/HHO-1 cells; lane 3, control PA317 retroviral packaging cells; lane 4, PA317/LXSN cells; lane 5, NIH 3T3/LXSN cells; lanes 6 and 7, NIH 3T3/HHO-1 cells; lane 8, control NIH 3T3 cells. ( B ) Reverse transcription (RT)-PCR analysis of PT67 retroviral packaging cells transduced with different retroviral vectors. Lanes 1 and 6, PT67 cells; lanes 2 and 7, PT67/LXSN cells; lanes 3 and 8, PT67/LSN-HOP cells; lanes 4 and 9, PT67/LSN-HOP-HHO-1 cells; Lanes 5 and 10, PT67/LSN-HOP-HHO-1-AS cells. m, Hin dIII-digested λ DNA marker; M, 100-bp DNA ruler; neo r , RT-PCR products of neomycin-resistance gene; HO-1, human HO-1 RT-PCR products. ( C ) Detection of human (h)HO-1 and G3PDH transcripts by Northern blot analysis in RLMV cells transduced with retroviral vectors LSN-HHO-1 or LXSN. Lanes 1, 2, and 5, RLMV cells transduced with LSN-HHO-1; lane 3, control RLMV cells; lane 4, RLMV cells transduced with LXSN.

Techniques Used: Northern Blot, Transduction, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Marker

37) Product Images from "Streamlining Homogeneous Glycoprotein Production for Biophysical and Structural Applications by Targeted Cell Line Development"

Article Title: Streamlining Homogeneous Glycoprotein Production for Biophysical and Structural Applications by Targeted Cell Line Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027829

Exchange of GFP with RFP by RMCE. (A) Green fluorescence of a GFP-tagged master cell clone (green), a GFP-negative RFP subclone (red) and CHO Lec3.2.8.1 cells (grey). (B) Red fluorescence of an RFP subcell clone upon one-week (orange) and 5-week (red) cultivation, compared to the corresponding master cells (grey). (C) Verification of RMCE by PCR amplification of the FRT-flanked gene cassette (GFP 1.9 kb, RFP 2.7 kb) from chromosomal DNA of cell lines derived from a master cell line with multi-copy transgenes. Lanes 1–5 and M represent 5 representative RFP positive subcell clones and the master cell line. Cell lines with incomplete exchange of integrated GFP gene copies were detected in lanes 1 and 3. (D) Expression strength of 7 GFP master cell clones and 2–4 corresponding RFP targeted subcell clones. Intracellular GFP (green) and RFP (red) fluorescence was measured by flow cytometry. GFP fluorescence was scaled down by a factor of 10.
Figure Legend Snippet: Exchange of GFP with RFP by RMCE. (A) Green fluorescence of a GFP-tagged master cell clone (green), a GFP-negative RFP subclone (red) and CHO Lec3.2.8.1 cells (grey). (B) Red fluorescence of an RFP subcell clone upon one-week (orange) and 5-week (red) cultivation, compared to the corresponding master cells (grey). (C) Verification of RMCE by PCR amplification of the FRT-flanked gene cassette (GFP 1.9 kb, RFP 2.7 kb) from chromosomal DNA of cell lines derived from a master cell line with multi-copy transgenes. Lanes 1–5 and M represent 5 representative RFP positive subcell clones and the master cell line. Cell lines with incomplete exchange of integrated GFP gene copies were detected in lanes 1 and 3. (D) Expression strength of 7 GFP master cell clones and 2–4 corresponding RFP targeted subcell clones. Intracellular GFP (green) and RFP (red) fluorescence was measured by flow cytometry. GFP fluorescence was scaled down by a factor of 10.

Techniques Used: Fluorescence, Polymerase Chain Reaction, Amplification, Derivative Assay, Clone Assay, Expressing, Flow Cytometry, Cytometry

Generation of master cell lines. (A) Selection of CHO Lec3.2.8.1 cells upon transfection with the GFP tagging vector pEF-FS-EGFP-dneo. One week post transfection, the top 2.6% fluorescent cells were isolated. Of these cells, the top 11% fluorescent cells were isolated as single cells one week later. The fluorescence profile of a representative cell clone is shown. (B) Fluorescence profile of a representative tagged cell clone in comparison to parental CHO Lec3.2.8.1 cells (marked with ‘C’). The GFP fluorescence of the tagged cells was observed over 12 weeks without measuring a reduction in fluorescence strength. (C) Southern blot analysis of integrated tagging vector copy numbers in six potential master cell clones. Genomic DNA was digested by BamHI, blotted and probed for GFP. Multiple bands indicate integration at multiple chromosomal sites. (D) PCR test for concatemers in four potential master cell clones. Primers are marked by horizontal arrows in panel C. PCR products were obtained only in the presence of tandem repeats. Cones = FRT sites (dark = F3, light = wild type).
Figure Legend Snippet: Generation of master cell lines. (A) Selection of CHO Lec3.2.8.1 cells upon transfection with the GFP tagging vector pEF-FS-EGFP-dneo. One week post transfection, the top 2.6% fluorescent cells were isolated. Of these cells, the top 11% fluorescent cells were isolated as single cells one week later. The fluorescence profile of a representative cell clone is shown. (B) Fluorescence profile of a representative tagged cell clone in comparison to parental CHO Lec3.2.8.1 cells (marked with ‘C’). The GFP fluorescence of the tagged cells was observed over 12 weeks without measuring a reduction in fluorescence strength. (C) Southern blot analysis of integrated tagging vector copy numbers in six potential master cell clones. Genomic DNA was digested by BamHI, blotted and probed for GFP. Multiple bands indicate integration at multiple chromosomal sites. (D) PCR test for concatemers in four potential master cell clones. Primers are marked by horizontal arrows in panel C. PCR products were obtained only in the presence of tandem repeats. Cones = FRT sites (dark = F3, light = wild type).

Techniques Used: Selection, Transfection, Plasmid Preparation, Isolation, Fluorescence, Southern Blot, Clone Assay, Polymerase Chain Reaction

38) Product Images from "The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I 1 Transcript Encoding a Subunit of Mitochondrial Complex I 1 [W] Transcript Encoding a Subunit of Mitochondrial Complex I 1 [W] [OPEN]"

Article Title: The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I 1 Transcript Encoding a Subunit of Mitochondrial Complex I 1 [W] Transcript Encoding a Subunit of Mitochondrial Complex I 1 [W] [OPEN]

Journal: Plant Physiology

doi: 10.1104/pp.114.244616

Detailed analysis of nad5 splicing. A, Diagram of the splicing events necessary for generating a translatable nad5 -PCR of the mature and unspliced mRNAs of individual nad5 exons in tang2
Figure Legend Snippet: Detailed analysis of nad5 splicing. A, Diagram of the splicing events necessary for generating a translatable nad5 -PCR of the mature and unspliced mRNAs of individual nad5 exons in tang2

Techniques Used: Polymerase Chain Reaction

-PCR is shown for nad1 , nad2 , nad3 , nad4 , nad5 , nad6 , nad7 , and nad9 transcripts in tang2 , tang2COM plants (A) and in otp439 , otp439COM -PCR of intron-containing mitochondrial
Figure Legend Snippet: -PCR is shown for nad1 , nad2 , nad3 , nad4 , nad5 , nad6 , nad7 , and nad9 transcripts in tang2 , tang2COM plants (A) and in otp439 , otp439COM -PCR of intron-containing mitochondrial

Techniques Used: Polymerase Chain Reaction

39) Product Images from "Generation and characterization of influenza A viruses with altered polymerase fidelity"

Article Title: Generation and characterization of influenza A viruses with altered polymerase fidelity

Journal: Nature communications

doi: 10.1038/ncomms5794

Replication kinetics of wild-type and PB1-V43I viruses under competition or suboptimal temperatures (a) One-step growth kinetics of wild-type and PB1-V43I H3N2 and H5N1 viruses using MOI 1–2 TCID 50 per cell in MDCK cells. Viral supernatants were collected every 2h post-infection and viral titres (mean ± SD log 10 TCID 50 per mL) from triplicates were shown. The replication kinetics has been repeated three times for the H3N2 wild-type and PB1-V43I viruses and once for the H5N1 wild-type and PB1-V43I viruses. (b) Competitive replication of wild-type and PB1-V43I mutant viruses in vitro . Wild-type and PB1-V43I viruses were premixed at different ratios prior to infection of MDCK cells. To determine the actual ratio in both the premixed (inoculum) and the viral supernatant after incubation for 2 days (progeny virus), clonal sequencing was performed to determine the ratio between Wuhan95 wild-type and PB1-V43I viruses. (c) As an alternative, in a separate experiment, instead of clonal sequencing, plaque assay was performed for the inoculums and passage-one viral supernatant after incubation for 2 days. Then, 32 clones were picked for each of the three inoculums and three corresponding passage-one viral cultures for viral RNA isolation and RT-PCR of the PB1 gene region that can distinguish wild-type virus from its V43I mutant counterpart. The actual ratio in the inoculums and the viral supernatant was shown in the same fashion. (d) Mean plaque sizes formed by the Wuhan95 wild-type and PB1-V43I viruses incubated at different temperatures. Wild-type or PB1-V43I mutant viruses were used to infect MDCK monolayers in triplicates and incubated under 0.5% agar overlay for 48 hours at 33°C, 37°C, or 39°C. The experiments were repeated independently twice and one representative result was shown. (e) Picture taken for the one representative plaque assay experiment performed for wild-type and V43I mutant viruses at the three temperatures. (f) Growth curve of wild-type and V43I mutant Wuhan95 viruses performed at 37°C, (g) 33°C, and (h) 39°C. For (f) to (h), viral titres (mean±SD log 10 TCID 50 per mL) from quadruplicated wells were shown. Three independent experiments were performed, with one representative experiment being displayed. P-values were based on Two-way ANOVA test with bonferroni post-tests. N.S., not statistically significant.
Figure Legend Snippet: Replication kinetics of wild-type and PB1-V43I viruses under competition or suboptimal temperatures (a) One-step growth kinetics of wild-type and PB1-V43I H3N2 and H5N1 viruses using MOI 1–2 TCID 50 per cell in MDCK cells. Viral supernatants were collected every 2h post-infection and viral titres (mean ± SD log 10 TCID 50 per mL) from triplicates were shown. The replication kinetics has been repeated three times for the H3N2 wild-type and PB1-V43I viruses and once for the H5N1 wild-type and PB1-V43I viruses. (b) Competitive replication of wild-type and PB1-V43I mutant viruses in vitro . Wild-type and PB1-V43I viruses were premixed at different ratios prior to infection of MDCK cells. To determine the actual ratio in both the premixed (inoculum) and the viral supernatant after incubation for 2 days (progeny virus), clonal sequencing was performed to determine the ratio between Wuhan95 wild-type and PB1-V43I viruses. (c) As an alternative, in a separate experiment, instead of clonal sequencing, plaque assay was performed for the inoculums and passage-one viral supernatant after incubation for 2 days. Then, 32 clones were picked for each of the three inoculums and three corresponding passage-one viral cultures for viral RNA isolation and RT-PCR of the PB1 gene region that can distinguish wild-type virus from its V43I mutant counterpart. The actual ratio in the inoculums and the viral supernatant was shown in the same fashion. (d) Mean plaque sizes formed by the Wuhan95 wild-type and PB1-V43I viruses incubated at different temperatures. Wild-type or PB1-V43I mutant viruses were used to infect MDCK monolayers in triplicates and incubated under 0.5% agar overlay for 48 hours at 33°C, 37°C, or 39°C. The experiments were repeated independently twice and one representative result was shown. (e) Picture taken for the one representative plaque assay experiment performed for wild-type and V43I mutant viruses at the three temperatures. (f) Growth curve of wild-type and V43I mutant Wuhan95 viruses performed at 37°C, (g) 33°C, and (h) 39°C. For (f) to (h), viral titres (mean±SD log 10 TCID 50 per mL) from quadruplicated wells were shown. Three independent experiments were performed, with one representative experiment being displayed. P-values were based on Two-way ANOVA test with bonferroni post-tests. N.S., not statistically significant.

Techniques Used: Infection, Mutagenesis, In Vitro, Incubation, Sequencing, Plaque Assay, Clone Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

40) Product Images from "Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone"

Article Title: Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone

Journal: mSphere

doi: 10.1128/mSphere.00246-16

Illustrations of ZIKV genome and plasmid. (A) Organization of the ZIKV single-stranded RNA genome showing the 5′ cap and positions of the mature viral proteins inside the single open reading frame. (B) Organization of the pCDNA6.2 ATCCMR766 Intron3127 HDVr plasmid carrying the cDNA of the 1947 Uganda MR766 ZIKV genome under the transcriptional control of the CMV promoter. A ribozyme (HDVr) is positioned to trim the RNA to have the ZIKV 3′ end. An intron was inserted after nucleotide 3127 of the viral sequence. The positions of the “GDD” NS5 polymerase active site [changed to GNN to create a replication-incompetent Pol(−) plasmid], beta-lactamase resistance gene (AmpR), and origin of replication (pUC ori) are also marked. (C) Schematic representation of the steps (described in detail in Materials and Methods) used to create the above plasmid involved cloning synthetic DNA gBlocks or RT-PCR products derived from viral inoculum to progressively assemble the entire genome. Solid lines above step iii represent deletions, and octagons and triangles above step v represent the positions of nonsense and frameshift mutations, respectively, obtained when cloning each of these fragments. Asterisks indicate the mutant that was used in the next cloning step.
Figure Legend Snippet: Illustrations of ZIKV genome and plasmid. (A) Organization of the ZIKV single-stranded RNA genome showing the 5′ cap and positions of the mature viral proteins inside the single open reading frame. (B) Organization of the pCDNA6.2 ATCCMR766 Intron3127 HDVr plasmid carrying the cDNA of the 1947 Uganda MR766 ZIKV genome under the transcriptional control of the CMV promoter. A ribozyme (HDVr) is positioned to trim the RNA to have the ZIKV 3′ end. An intron was inserted after nucleotide 3127 of the viral sequence. The positions of the “GDD” NS5 polymerase active site [changed to GNN to create a replication-incompetent Pol(−) plasmid], beta-lactamase resistance gene (AmpR), and origin of replication (pUC ori) are also marked. (C) Schematic representation of the steps (described in detail in Materials and Methods) used to create the above plasmid involved cloning synthetic DNA gBlocks or RT-PCR products derived from viral inoculum to progressively assemble the entire genome. Solid lines above step iii represent deletions, and octagons and triangles above step v represent the positions of nonsense and frameshift mutations, respectively, obtained when cloning each of these fragments. Asterisks indicate the mutant that was used in the next cloning step.

Techniques Used: Plasmid Preparation, Sequencing, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Mutagenesis

41) Product Images from "Loss of Octarepeats in Two Processed Prion Pseudogenes in the Red Squirrel, Sciurus vulgaris"

Article Title: Loss of Octarepeats in Two Processed Prion Pseudogenes in the Red Squirrel, Sciurus vulgaris

Journal: Journal of Molecular Evolution

doi: 10.1007/s00239-010-9390-7

PCR amplification with PRNP -specific primers on samples of genomic DNA ( a – c ) and total brain cDNA ( d ) resolved on 1% agarose gels. In panel a , lanes 1 and 2 were amplified with the ‘short’ primer pair, all other lanes with the ‘long’ primer pair; in panels b , c and d the ‘long1’ primer pair was applied (see ‘ Materials and Methods ’). All PCR products were sequenced to determine the number of repeats. 2R, 3R and 5R correspond with the presence of 2, 3 and 5 (or 6 in tenrec) repeats. a Lanes 1 – 7 , red squirrels ( S. vulgaris ) from different localities in the Netherlands; lane 8 , human ( Homo sapiens ); lane 9 , black rhino ( Diceros bicornis ); lane 10 , Indian elephant (Elephas maximus); lane 11 , tenrec ( Tenrec ecaudatus ). b Lanes 1 – 6 , red squirrels ( S. vulgaris ) from Germany, Spain, Wales, Italy, Eastern Siberia and the Netherlands, respectively. c Lane 1 , grey squirrel ( S. carolinensis ); lane 2 , Japanese squirrel ( S. lis ). d cDNA from red squirrel ( S. vulgaris ) brain; +RT and −RT indicates the presence or the absence, respectively, of reverse transcriptase during cDNA synthesis
Figure Legend Snippet: PCR amplification with PRNP -specific primers on samples of genomic DNA ( a – c ) and total brain cDNA ( d ) resolved on 1% agarose gels. In panel a , lanes 1 and 2 were amplified with the ‘short’ primer pair, all other lanes with the ‘long’ primer pair; in panels b , c and d the ‘long1’ primer pair was applied (see ‘ Materials and Methods ’). All PCR products were sequenced to determine the number of repeats. 2R, 3R and 5R correspond with the presence of 2, 3 and 5 (or 6 in tenrec) repeats. a Lanes 1 – 7 , red squirrels ( S. vulgaris ) from different localities in the Netherlands; lane 8 , human ( Homo sapiens ); lane 9 , black rhino ( Diceros bicornis ); lane 10 , Indian elephant (Elephas maximus); lane 11 , tenrec ( Tenrec ecaudatus ). b Lanes 1 – 6 , red squirrels ( S. vulgaris ) from Germany, Spain, Wales, Italy, Eastern Siberia and the Netherlands, respectively. c Lane 1 , grey squirrel ( S. carolinensis ); lane 2 , Japanese squirrel ( S. lis ). d cDNA from red squirrel ( S. vulgaris ) brain; +RT and −RT indicates the presence or the absence, respectively, of reverse transcriptase during cDNA synthesis

Techniques Used: Polymerase Chain Reaction, Amplification

42) Product Images from "Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis"

Article Title: Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis

Journal: Infection and Immunity

doi: 10.1128/IAI.70.6.3234-3248.2002

RT-PCR analysis of IDO mRNA expression in dTHP-1 cells and MdM stimulated for 24 h with C. trachomatis -infected HeLa cell supernatants and in dTHP-1 cells cocultivated with infected HeLa cells for 24 and 48 h. The cDNAs were amplified with IDO (324 bp, top panel) and GAPDH (306 bp, bottom panel) primers for 35 and 22 PCR cycles, respectively. A total of 10 μl of PCR products was loaded on a 2% agarose gel as follows. dTHP-1 cells and MdM were incubated with supernatants from HeLa cells left uninfected (lanes 1 and 6, respectively), infected with serovar E (lanes 2 and 7, respectively) or serovar L2 (lanes 3 and 8, respectively), treated with RPMI alone (lanes 4 and 9, respectively) or E. coli LPS alone (lanes 5 and 10, respectively), or dTHP-1 cells were incubated in coculture for 24 and 48 h with uninfected HeLa cells (lanes 11 and 14, respectively), with serovar E-infected HeLa cells (lanes 12 and 15, respectively), or with serovar L2-infected HeLa cells (lanes 13 and 16, respectively). A negative control for amplification (lane 17) wherein DNA was omitted and a positive control (lane 18) consisting of cDNA from HeLa cells exposed to rhIFN-γ (10 ng/ml for 12 h) were also included in the analysis.
Figure Legend Snippet: RT-PCR analysis of IDO mRNA expression in dTHP-1 cells and MdM stimulated for 24 h with C. trachomatis -infected HeLa cell supernatants and in dTHP-1 cells cocultivated with infected HeLa cells for 24 and 48 h. The cDNAs were amplified with IDO (324 bp, top panel) and GAPDH (306 bp, bottom panel) primers for 35 and 22 PCR cycles, respectively. A total of 10 μl of PCR products was loaded on a 2% agarose gel as follows. dTHP-1 cells and MdM were incubated with supernatants from HeLa cells left uninfected (lanes 1 and 6, respectively), infected with serovar E (lanes 2 and 7, respectively) or serovar L2 (lanes 3 and 8, respectively), treated with RPMI alone (lanes 4 and 9, respectively) or E. coli LPS alone (lanes 5 and 10, respectively), or dTHP-1 cells were incubated in coculture for 24 and 48 h with uninfected HeLa cells (lanes 11 and 14, respectively), with serovar E-infected HeLa cells (lanes 12 and 15, respectively), or with serovar L2-infected HeLa cells (lanes 13 and 16, respectively). A negative control for amplification (lane 17) wherein DNA was omitted and a positive control (lane 18) consisting of cDNA from HeLa cells exposed to rhIFN-γ (10 ng/ml for 12 h) were also included in the analysis.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Incubation, Negative Control, Positive Control

43) Product Images from "CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose"

Article Title: CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose

Journal: Plant Biotechnology Journal

doi: 10.1111/pbi.12981

Sequencing and Western blot analysis for the T 2 lines X‐KO #34‐4‐4 and F‐KO #6‐10‐6 and the F 2 line FX‐KO #20‐4. (a) Mutations in XylT 1 and 2 of X‐KO #34‐4‐4 as identified by Sanger sequencing of PCR amplicons ( XylT 1) and TOPO‐cloned PCR products ( XylT 2). The corresponding wild‐type sequence is shown above, the gRNA target sequences are indicated by coloured boxes, and the PAM sequences are shown in bold. (b) Mutations in FucT 1, 2, 3 and 4 of F‐KO #6‐10‐6. The PAM‐distal mismatch between the gRNA and Fuc T 3 and 4 is highlighted in yellow. (c) Mutations in FucT 1‐4 and XylT 1 and 2 of FX‐KO #20‐4. Five of six genes carry homozygous mutations, with the exception of FucT 3 which has biallelic mutations. (d) Western blot of FX‐KO #20‐4 in comparison to N. benthamiana wild type, F‐KO #6‐10‐6 and X‐KO #34‐4‐4. ~10 µg of total soluble protein were loaded for each sample. Anti‐fucose blot: 1st antibody rabbit‐anti‐α1,3‐fucose (1 : 10 000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed). Anti‐xylose blot: 1st antibody rabbit‐anti‐β1,2‐xylose (1 : 5000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed).
Figure Legend Snippet: Sequencing and Western blot analysis for the T 2 lines X‐KO #34‐4‐4 and F‐KO #6‐10‐6 and the F 2 line FX‐KO #20‐4. (a) Mutations in XylT 1 and 2 of X‐KO #34‐4‐4 as identified by Sanger sequencing of PCR amplicons ( XylT 1) and TOPO‐cloned PCR products ( XylT 2). The corresponding wild‐type sequence is shown above, the gRNA target sequences are indicated by coloured boxes, and the PAM sequences are shown in bold. (b) Mutations in FucT 1, 2, 3 and 4 of F‐KO #6‐10‐6. The PAM‐distal mismatch between the gRNA and Fuc T 3 and 4 is highlighted in yellow. (c) Mutations in FucT 1‐4 and XylT 1 and 2 of FX‐KO #20‐4. Five of six genes carry homozygous mutations, with the exception of FucT 3 which has biallelic mutations. (d) Western blot of FX‐KO #20‐4 in comparison to N. benthamiana wild type, F‐KO #6‐10‐6 and X‐KO #34‐4‐4. ~10 µg of total soluble protein were loaded for each sample. Anti‐fucose blot: 1st antibody rabbit‐anti‐α1,3‐fucose (1 : 10 000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed). Anti‐xylose blot: 1st antibody rabbit‐anti‐β1,2‐xylose (1 : 5000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed).

Techniques Used: Sequencing, Western Blot, Polymerase Chain Reaction, Clone Assay

44) Product Images from "Balancing Selection Maintains a Form of ERAP2 that Undergoes Nonsense-Mediated Decay and Affects Antigen Presentation"

Article Title: Balancing Selection Maintains a Form of ERAP2 that Undergoes Nonsense-Mediated Decay and Affects Antigen Presentation

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1001157

Haplotype-specific splicing of ERAP2 . A, The genomic organization of the human chromosome 5q15 region containing ERAP1 and ERAP2 is included at the top. The two haplotype-specific ERAP2 spliced forms are shown for Haplotype A (in blue) and Haplotype B (in purple). The different alleles of rs2248374 are shown as a blue or purple base position, respectively. The red boxes represent the premature stop codons in the Haplotype B mRNA. B, PCR amplification of cDNA across the exon 10 splice junction (see Materials and Methods ) from the indicated 16 LCLs, with the haplotype status of each cell indicated as homozygote (AA or BB) or heterozygote (AB). A negative control PCR, with no DNA template, was also performed (water).
Figure Legend Snippet: Haplotype-specific splicing of ERAP2 . A, The genomic organization of the human chromosome 5q15 region containing ERAP1 and ERAP2 is included at the top. The two haplotype-specific ERAP2 spliced forms are shown for Haplotype A (in blue) and Haplotype B (in purple). The different alleles of rs2248374 are shown as a blue or purple base position, respectively. The red boxes represent the premature stop codons in the Haplotype B mRNA. B, PCR amplification of cDNA across the exon 10 splice junction (see Materials and Methods ) from the indicated 16 LCLs, with the haplotype status of each cell indicated as homozygote (AA or BB) or heterozygote (AB). A negative control PCR, with no DNA template, was also performed (water).

Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control

45) Product Images from "Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator"

Article Title: Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001255

DNAJC14 confers resistance to YFV-induced cell death. (A) Photographs 7 d after YFV challenge (moi = 1) of SW13 cells transduced with Round 3 of the selected lentiviral cDNA constructs compared to cells transduced with V1-GFP vector control. (B) The cells transduced with the Round 3 lentivirus pool and surviving YFV infection (Rd 3) were expanded and reinfected with YFV at the indicated moi. Crystal violet staining was performed 3 d later. Cells transduced with vector alone serve as a control (V1-GFP). (C) DNA was isolated from naïve SW13 or Round 3 (Rd 3) cells, and the lentiviral insert amplified by PCR. The major band was identified as encoding a truncated hamster DNAJC14. Sizes of the DNA markers (kb) are indicated to the left. (D) A schematic of human DNAJC14 is shown, with the putative transmembrane (TM) domains (gray), J domain (red) with conserved HPD sequence, zinc finger motifs (blue) and Jiv90 domain (orange) indicated. A schematic of the isolated hamster clone, showing homology to amino acids 305 to 702 of human DNAJC14, is shown below.
Figure Legend Snippet: DNAJC14 confers resistance to YFV-induced cell death. (A) Photographs 7 d after YFV challenge (moi = 1) of SW13 cells transduced with Round 3 of the selected lentiviral cDNA constructs compared to cells transduced with V1-GFP vector control. (B) The cells transduced with the Round 3 lentivirus pool and surviving YFV infection (Rd 3) were expanded and reinfected with YFV at the indicated moi. Crystal violet staining was performed 3 d later. Cells transduced with vector alone serve as a control (V1-GFP). (C) DNA was isolated from naïve SW13 or Round 3 (Rd 3) cells, and the lentiviral insert amplified by PCR. The major band was identified as encoding a truncated hamster DNAJC14. Sizes of the DNA markers (kb) are indicated to the left. (D) A schematic of human DNAJC14 is shown, with the putative transmembrane (TM) domains (gray), J domain (red) with conserved HPD sequence, zinc finger motifs (blue) and Jiv90 domain (orange) indicated. A schematic of the isolated hamster clone, showing homology to amino acids 305 to 702 of human DNAJC14, is shown below.

Techniques Used: Transduction, Construct, Plasmid Preparation, Infection, Staining, Isolation, Amplification, Polymerase Chain Reaction, Sequencing

46) Product Images from "Global Analysis of the HrpL Regulon in the Plant Pathogen Pseudomonas syringae pv. tomato DC3000 Reveals New Regulon Members with Diverse Functions"

Article Title: Global Analysis of the HrpL Regulon in the Plant Pathogen Pseudomonas syringae pv. tomato DC3000 Reveals New Regulon Members with Diverse Functions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0106115

Validation of new hrp promoters. (A). ChIP_qPCR experiments to test enrichment of DNA fragments at putative HrpL binding sites. Values for each gene were normalized to results for gyrA (DNA gyrase subunit A). gap-1 (glyceraldehyde 3-phosphate dehydrogenase, type I), not predicted to be HrpL-regulated, was used as a negative control. All fold changes above the expression value for gyrA are classified as enriched (above the horizontal line). (B). Induction of cloned hrp promoter- gfp fusions. Induction was measured by relative fluorescence normalized by OD 600 (GFP fluorescence/OD) in hrp -inducing and hrp -repressing conditions. The hrp promoter:: gfp fusion constructs were expressed in the DC3000 Δ pvsA siderophore mutant. The promoter trap vector without a promoter insert was used as a negative control (NC). GFP was measured using a Synergy 2 plate reader (Biotech) with excitation from 475 to 495 nm and emission from 506 to 526 nm. OD was measured at 600 nm using the same plate reader. A kinetics reading procedure was used, and a single data point at 5 hours was plotted for all strains, which is the time at which they show a peak value. (C). qRT-PCR analysis showing HrpL-dependent differential expression of transcripts downstream from hrp promoters in WT DC3000 and ΔhrpL strains. The relative fold change was measured after 1.5 hours on MG supplemented with iron (50 µM final concentration) normalized to gyrA. For determination of the relative expression, expression of each gene in the ΔhrpL mutant was set to 1. Expression of each gene in the WT strain was then normalized to the corresponding gene in the ΔhrpL mutant. All data points are the averages of 3 replicates with standard deviations.
Figure Legend Snippet: Validation of new hrp promoters. (A). ChIP_qPCR experiments to test enrichment of DNA fragments at putative HrpL binding sites. Values for each gene were normalized to results for gyrA (DNA gyrase subunit A). gap-1 (glyceraldehyde 3-phosphate dehydrogenase, type I), not predicted to be HrpL-regulated, was used as a negative control. All fold changes above the expression value for gyrA are classified as enriched (above the horizontal line). (B). Induction of cloned hrp promoter- gfp fusions. Induction was measured by relative fluorescence normalized by OD 600 (GFP fluorescence/OD) in hrp -inducing and hrp -repressing conditions. The hrp promoter:: gfp fusion constructs were expressed in the DC3000 Δ pvsA siderophore mutant. The promoter trap vector without a promoter insert was used as a negative control (NC). GFP was measured using a Synergy 2 plate reader (Biotech) with excitation from 475 to 495 nm and emission from 506 to 526 nm. OD was measured at 600 nm using the same plate reader. A kinetics reading procedure was used, and a single data point at 5 hours was plotted for all strains, which is the time at which they show a peak value. (C). qRT-PCR analysis showing HrpL-dependent differential expression of transcripts downstream from hrp promoters in WT DC3000 and ΔhrpL strains. The relative fold change was measured after 1.5 hours on MG supplemented with iron (50 µM final concentration) normalized to gyrA. For determination of the relative expression, expression of each gene in the ΔhrpL mutant was set to 1. Expression of each gene in the WT strain was then normalized to the corresponding gene in the ΔhrpL mutant. All data points are the averages of 3 replicates with standard deviations.

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control, Expressing, Clone Assay, Fluorescence, Construct, Mutagenesis, Plasmid Preparation, Quantitative RT-PCR, Concentration Assay

47) Product Images from "Increased tumour necrosis factor ? production in mesenteric lymph nodes of cirrhotic patients with ascites"

Article Title: Increased tumour necrosis factor ? production in mesenteric lymph nodes of cirrhotic patients with ascites

Journal: Gut

doi:

Agarose gel electrophoresis of competitive polymerase chain reaction for tumour necrosis factor α (TNF) mRNA. From left to right: molecular weight marker, blank amplification with the internal calibration standard (ICS) band, competitive amplification of ICS, and TNF cDNA at two different dilutions of cDNA.
Figure Legend Snippet: Agarose gel electrophoresis of competitive polymerase chain reaction for tumour necrosis factor α (TNF) mRNA. From left to right: molecular weight marker, blank amplification with the internal calibration standard (ICS) band, competitive amplification of ICS, and TNF cDNA at two different dilutions of cDNA.

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight, Marker, Amplification

48) Product Images from "A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides"

Article Title: A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002042

Phylogenetic relationships of the HDMs. (A) A bootstrapped (1000 trials) neighbour-joining phylogenetic tree showing the evolutionary relationship of HDM cDNA sequences from medically-important trematode pathogens. Numbers represent bootstrap values (given as percentages) for a particular node, and values greater than 65% are shown. The tree is rooted to human CAP18 (accession number NM_004345). Three major clades are shown corresponding to the Sm16-like molecules, the schistosome HDMs and HDMs from Fasciola and the Asian flukes. (B) Primary sequence alignment of selected members of the HDM clades. Conserved residues that contribute to the hydrophobic face of the amphipathic helix are shaded in grey. (C) Top panel. RT-PCR analysis of FhHDM-1 expression in F. hepatica newly excysted juveniles (NEJ), 21-day immature flukes (21d) and adult worms (Adult). Amplification of constitutively expressed F. hepatica β-actin was performed as a positive control. Samples were separated by agarose gel electrophoresis and stained with ethidium bromide. Bottom panel. Immunogenicity of FhHDM-1 in F. hepatica -infected sheep. Pre-infection sera (Pre) and samples taken 4, 8, 12 and 16 weeks post-infection were analysed by ELISA and Western blot using an anti-FhHDM-1 antibody. Specific antibody responses were detected at week 4 with immunoblot staining stronger at weeks 8 and 12 after infection.
Figure Legend Snippet: Phylogenetic relationships of the HDMs. (A) A bootstrapped (1000 trials) neighbour-joining phylogenetic tree showing the evolutionary relationship of HDM cDNA sequences from medically-important trematode pathogens. Numbers represent bootstrap values (given as percentages) for a particular node, and values greater than 65% are shown. The tree is rooted to human CAP18 (accession number NM_004345). Three major clades are shown corresponding to the Sm16-like molecules, the schistosome HDMs and HDMs from Fasciola and the Asian flukes. (B) Primary sequence alignment of selected members of the HDM clades. Conserved residues that contribute to the hydrophobic face of the amphipathic helix are shaded in grey. (C) Top panel. RT-PCR analysis of FhHDM-1 expression in F. hepatica newly excysted juveniles (NEJ), 21-day immature flukes (21d) and adult worms (Adult). Amplification of constitutively expressed F. hepatica β-actin was performed as a positive control. Samples were separated by agarose gel electrophoresis and stained with ethidium bromide. Bottom panel. Immunogenicity of FhHDM-1 in F. hepatica -infected sheep. Pre-infection sera (Pre) and samples taken 4, 8, 12 and 16 weeks post-infection were analysed by ELISA and Western blot using an anti-FhHDM-1 antibody. Specific antibody responses were detected at week 4 with immunoblot staining stronger at weeks 8 and 12 after infection.

Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Positive Control, Agarose Gel Electrophoresis, Staining, Infection, Enzyme-linked Immunosorbent Assay, Western Blot

49) Product Images from "The Retinol Binding Protein Receptor 2 (Rbpr2) is required for Photoreceptor Outer Segment Morphogenesis and Visual Function in Zebrafish"

Article Title: The Retinol Binding Protein Receptor 2 (Rbpr2) is required for Photoreceptor Outer Segment Morphogenesis and Visual Function in Zebrafish

Journal: Scientific Reports

doi: 10.1038/s41598-017-16498-9

Downregulation of retinoid signaling regulated genes in rbpr2 musc97 mutants. ( A ) Retina-specific gene expression were compared by qRT-PCR using equal amounts of total RNA from heads of WT (black bars) and rbpr2 musc97 mutants (grey bars), at 5.5 dpf. aldh1a2 , dhrs3a , cyp26a1 , lrat and rpe65 mRNA expression were normalized to 18 S ribosomal RNA. mRNA expression values of genes in WT animals were set to 1, and difference in gene expression between the two genotypes are shown as relative fold change normalized to endogenous 18 S RNA. *p
Figure Legend Snippet: Downregulation of retinoid signaling regulated genes in rbpr2 musc97 mutants. ( A ) Retina-specific gene expression were compared by qRT-PCR using equal amounts of total RNA from heads of WT (black bars) and rbpr2 musc97 mutants (grey bars), at 5.5 dpf. aldh1a2 , dhrs3a , cyp26a1 , lrat and rpe65 mRNA expression were normalized to 18 S ribosomal RNA. mRNA expression values of genes in WT animals were set to 1, and difference in gene expression between the two genotypes are shown as relative fold change normalized to endogenous 18 S RNA. *p

Techniques Used: Expressing, Quantitative RT-PCR

50) Product Images from "Impaired activity-dependent FMRP translation and enhanced mGluR-dependent LTD in Fragile X premutation mice"

Article Title: Impaired activity-dependent FMRP translation and enhanced mGluR-dependent LTD in Fragile X premutation mice

Journal: Human Molecular Genetics

doi: 10.1093/hmg/dds525

Elevated cortical Fmr1 mRNA and decreased Fragile X mental retardation protein (FMRP) in the fragile X premutation mouse. ( A ) PCR genotyping of CGG KI male mice and WT littermates showing the expanded CGG repeat. KI band corresponds to ∼120 repeats; WT band corresponds to 8 CGG repeats. ( B ) Fmr1 mRNA levels in the cortex of p28–37 fragile X premutation male mice by qPCR using two different sets of primers against Fmr1 . The bar graph summarizes three experiments, n = 5. ( C ) Representative immunoblot to FMRP (1C3 1:1000) in p28–37 male mouse cortices from the indicated genotypes. Below: Summary of three experiments. Mean (±SEM) cortical FMRP in 1-month-old (p28–38; n = 5) and 6-month-old (p177–181; n = 3) CGG KI mice is decreased compared with littermate controls. The relative decrease between genotypes is greater in older animals. ( D ) Representative immunoblot against FMRP (17722 1:1000) in hippocampi of p35 CGG KI animals compared with WT littermate controls. Below: Mean (±SEM) hippocampal FMRP in p35–p60 male CGG KI mice compared with WT littermate controls. n = 5. ( E ) Translational efficiency of cortical Fmr1 RNA expressed as the ratio of FMRP to Fmr1 RNA levels in each individual animal, plotted on log 10 scale; n = 5. * P
Figure Legend Snippet: Elevated cortical Fmr1 mRNA and decreased Fragile X mental retardation protein (FMRP) in the fragile X premutation mouse. ( A ) PCR genotyping of CGG KI male mice and WT littermates showing the expanded CGG repeat. KI band corresponds to ∼120 repeats; WT band corresponds to 8 CGG repeats. ( B ) Fmr1 mRNA levels in the cortex of p28–37 fragile X premutation male mice by qPCR using two different sets of primers against Fmr1 . The bar graph summarizes three experiments, n = 5. ( C ) Representative immunoblot to FMRP (1C3 1:1000) in p28–37 male mouse cortices from the indicated genotypes. Below: Summary of three experiments. Mean (±SEM) cortical FMRP in 1-month-old (p28–38; n = 5) and 6-month-old (p177–181; n = 3) CGG KI mice is decreased compared with littermate controls. The relative decrease between genotypes is greater in older animals. ( D ) Representative immunoblot against FMRP (17722 1:1000) in hippocampi of p35 CGG KI animals compared with WT littermate controls. Below: Mean (±SEM) hippocampal FMRP in p35–p60 male CGG KI mice compared with WT littermate controls. n = 5. ( E ) Translational efficiency of cortical Fmr1 RNA expressed as the ratio of FMRP to Fmr1 RNA levels in each individual animal, plotted on log 10 scale; n = 5. * P

Techniques Used: Polymerase Chain Reaction, Mouse Assay, Real-time Polymerase Chain Reaction

51) Product Images from "Sequence Variations of Full-Length Hepatitis B Virus Genomes in Chinese Patients with HBsAg-Negative Hepatitis B Infection"

Article Title: Sequence Variations of Full-Length Hepatitis B Virus Genomes in Chinese Patients with HBsAg-Negative Hepatitis B Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099028

Rolling circle amplification (RCA) of full-length HBV genome. The number of initial HBV DNA template present in each RCA reaction is shown at the top. M, molecular weight marker; H2O, negative control. (A) The high-molecular-weight raw RCA products containing multiple copies of the initial HBV template. (B) The full-length HBV genomes recovered by restriction enzyme, Spe I digestion. (C) Full-length HBV genome amplified by using the RCA products as PCR template.
Figure Legend Snippet: Rolling circle amplification (RCA) of full-length HBV genome. The number of initial HBV DNA template present in each RCA reaction is shown at the top. M, molecular weight marker; H2O, negative control. (A) The high-molecular-weight raw RCA products containing multiple copies of the initial HBV template. (B) The full-length HBV genomes recovered by restriction enzyme, Spe I digestion. (C) Full-length HBV genome amplified by using the RCA products as PCR template.

Techniques Used: Amplification, Molecular Weight, Marker, Negative Control, Polymerase Chain Reaction

52) Product Images from "Biochemical and molecular characterization of 3-Methylcrotonylglycinuria in an Italian asymptomatic girl"

Article Title: Biochemical and molecular characterization of 3-Methylcrotonylglycinuria in an Italian asymptomatic girl

Journal: Genetics and Molecular Biology

doi: 10.1590/1678-4685-GMB-2017-0093

Minigene construct and RT-PCR results obtained in expression studies. (A) The minigene construct (pMGene) used in the study: a DNA fragment of approximately 1 kb was directly amplified from the genomic DNA of the patient and cloned into the pMGene as decribed in the Results section. All clones were sequenced, and a wild-type and a mutated clone were used for expression experiments. (B and C) The splicing pattern, evaluated by RT-PCR and sequence analysis of mRNA extracted from cell lines transfected with WT or mutated minigene constructs. Lane 1: molecular weight marker, lane 2: RT-PCR of RNA obtained using the mutant clone; lane 3: RT-PCR of RNA obtained from the normal clone.
Figure Legend Snippet: Minigene construct and RT-PCR results obtained in expression studies. (A) The minigene construct (pMGene) used in the study: a DNA fragment of approximately 1 kb was directly amplified from the genomic DNA of the patient and cloned into the pMGene as decribed in the Results section. All clones were sequenced, and a wild-type and a mutated clone were used for expression experiments. (B and C) The splicing pattern, evaluated by RT-PCR and sequence analysis of mRNA extracted from cell lines transfected with WT or mutated minigene constructs. Lane 1: molecular weight marker, lane 2: RT-PCR of RNA obtained using the mutant clone; lane 3: RT-PCR of RNA obtained from the normal clone.

Techniques Used: Construct, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Clone Assay, Sequencing, Transfection, Molecular Weight, Marker, Mutagenesis

53) Product Images from "Biochemical and molecular characterization of 3-Methylcrotonylglycinuria in an Italian asymptomatic girl"

Article Title: Biochemical and molecular characterization of 3-Methylcrotonylglycinuria in an Italian asymptomatic girl

Journal: Genetics and Molecular Biology

doi: 10.1590/1678-4685-GMB-2017-0093

Minigene construct and RT-PCR results obtained in expression studies. (A) The minigene construct (pMGene) used in the study: a DNA fragment of approximately 1 kb was directly amplified from the genomic DNA of the patient and cloned into the pMGene as decribed in the Results section. All clones were sequenced, and a wild-type and a mutated clone were used for expression experiments. (B and C) The splicing pattern, evaluated by RT-PCR and sequence analysis of mRNA extracted from cell lines transfected with WT or mutated minigene constructs. Lane 1: molecular weight marker, lane 2: RT-PCR of RNA obtained using the mutant clone; lane 3: RT-PCR of RNA obtained from the normal clone.
Figure Legend Snippet: Minigene construct and RT-PCR results obtained in expression studies. (A) The minigene construct (pMGene) used in the study: a DNA fragment of approximately 1 kb was directly amplified from the genomic DNA of the patient and cloned into the pMGene as decribed in the Results section. All clones were sequenced, and a wild-type and a mutated clone were used for expression experiments. (B and C) The splicing pattern, evaluated by RT-PCR and sequence analysis of mRNA extracted from cell lines transfected with WT or mutated minigene constructs. Lane 1: molecular weight marker, lane 2: RT-PCR of RNA obtained using the mutant clone; lane 3: RT-PCR of RNA obtained from the normal clone.

Techniques Used: Construct, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Clone Assay, Sequencing, Transfection, Molecular Weight, Marker, Mutagenesis

54) Product Images from "Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas aeruginosa With Bronchial Epithelial Cells"

Article Title: Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas aeruginosa With Bronchial Epithelial Cells

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.03018

Analysis of pqsA , pqsE , mexG, and lecA transcription in WT and Δ pqsA Calu-3-ALI-infected cultures in the presence and absence of exogenous PQS. Expression of pqsA , pqsE , mexG, and lecA during infection of Calu-3-ALI cultures with PAO1 and Δ pqsA PAO1 at 3 hpi was assessed by RT-PCR. Calu-3-ALI cultures exposed to PQS (40 μM) or DMSO were infected at MOI 50 and total RNA prepared at 3 hpi. A 250-bp DNA region within the pqsA gene and a 200-bp DNA region within pqsE, oprL , mexG, and lecA genes were amplified from PAO1 genomic DNA (positive control); 1, cDNA; 2, corresponding RNA (negative control). L, 50 bp DNA ladder. Data are representative from 2 independent experiments.
Figure Legend Snippet: Analysis of pqsA , pqsE , mexG, and lecA transcription in WT and Δ pqsA Calu-3-ALI-infected cultures in the presence and absence of exogenous PQS. Expression of pqsA , pqsE , mexG, and lecA during infection of Calu-3-ALI cultures with PAO1 and Δ pqsA PAO1 at 3 hpi was assessed by RT-PCR. Calu-3-ALI cultures exposed to PQS (40 μM) or DMSO were infected at MOI 50 and total RNA prepared at 3 hpi. A 250-bp DNA region within the pqsA gene and a 200-bp DNA region within pqsE, oprL , mexG, and lecA genes were amplified from PAO1 genomic DNA (positive control); 1, cDNA; 2, corresponding RNA (negative control). L, 50 bp DNA ladder. Data are representative from 2 independent experiments.

Techniques Used: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Negative Control

55) Product Images from "Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas aeruginosa With Bronchial Epithelial Cells"

Article Title: Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas aeruginosa With Bronchial Epithelial Cells

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.03018

Analysis of pqsA , pqsE , mexG, and lecA transcription in WT and Δ pqsA Calu-3-ALI-infected cultures in the presence and absence of exogenous PQS. Expression of pqsA , pqsE , mexG, and lecA during infection of Calu-3-ALI cultures with PAO1 and Δ pqsA PAO1 at 3 hpi was assessed by RT-PCR. Calu-3-ALI cultures exposed to PQS (40 μM) or DMSO were infected at MOI 50 and total RNA prepared at 3 hpi. A 250-bp DNA region within the pqsA gene and a 200-bp DNA region within pqsE, oprL , mexG, and lecA genes were amplified from PAO1 genomic DNA (positive control); 1, cDNA; 2, corresponding RNA (negative control). L, 50 bp DNA ladder. Data are representative from 2 independent experiments.
Figure Legend Snippet: Analysis of pqsA , pqsE , mexG, and lecA transcription in WT and Δ pqsA Calu-3-ALI-infected cultures in the presence and absence of exogenous PQS. Expression of pqsA , pqsE , mexG, and lecA during infection of Calu-3-ALI cultures with PAO1 and Δ pqsA PAO1 at 3 hpi was assessed by RT-PCR. Calu-3-ALI cultures exposed to PQS (40 μM) or DMSO were infected at MOI 50 and total RNA prepared at 3 hpi. A 250-bp DNA region within the pqsA gene and a 200-bp DNA region within pqsE, oprL , mexG, and lecA genes were amplified from PAO1 genomic DNA (positive control); 1, cDNA; 2, corresponding RNA (negative control). L, 50 bp DNA ladder. Data are representative from 2 independent experiments.

Techniques Used: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Negative Control

56) Product Images from "The Promoter of the pri-miR-375 Gene Directs Expression Selectively to the Endocrine Pancreas"

Article Title: The Promoter of the pri-miR-375 Gene Directs Expression Selectively to the Endocrine Pancreas

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005033

Mapping of transcription start site of miR-375 gene by 5′-RACE. A. DNase-treated total RNA from HIT cells transfected with the pGL3-375a construct was reverse transcribed, using a primer complementary to the luciferase sequence. The cDNA was poly-(dG) tailed and amplified by PCR using a poly-C primer and a nested primer complementary to block 4 (lane 1). Lane 2, no RT control; lane 3, no template control. Marker sizes are indicated. The location of the band in lane 1 is indicated by an arrow. B. Alignment of miR-375 upstream sequences (from −96 to +244, relative to transcription start site) of mouse, rat and human. The primer used for PCR amplification is located at the middle of block 4. The TATA sequence is marked by a box. The large arrow-head indicates the major transcription start site. Small arrow-heads indicate the start site of shorter species detected by 5′-RACE. C. Representation of the pGL3-375a construct. Conserved regions 1–4 are indicated. The luciferase gene is indicated as an unmarked open box. The arrow indicates the transcription start site revealed by 5′-RACE analysis. Numbers above the bar indicate location relative to the transcription start site.
Figure Legend Snippet: Mapping of transcription start site of miR-375 gene by 5′-RACE. A. DNase-treated total RNA from HIT cells transfected with the pGL3-375a construct was reverse transcribed, using a primer complementary to the luciferase sequence. The cDNA was poly-(dG) tailed and amplified by PCR using a poly-C primer and a nested primer complementary to block 4 (lane 1). Lane 2, no RT control; lane 3, no template control. Marker sizes are indicated. The location of the band in lane 1 is indicated by an arrow. B. Alignment of miR-375 upstream sequences (from −96 to +244, relative to transcription start site) of mouse, rat and human. The primer used for PCR amplification is located at the middle of block 4. The TATA sequence is marked by a box. The large arrow-head indicates the major transcription start site. Small arrow-heads indicate the start site of shorter species detected by 5′-RACE. C. Representation of the pGL3-375a construct. Conserved regions 1–4 are indicated. The luciferase gene is indicated as an unmarked open box. The arrow indicates the transcription start site revealed by 5′-RACE analysis. Numbers above the bar indicate location relative to the transcription start site.

Techniques Used: Transfection, Construct, Luciferase, Sequencing, Amplification, Polymerase Chain Reaction, Blocking Assay, Marker

57) Product Images from "The Role of Hepatocyte Hemojuvelin in the Regulation of Bone Morphogenic Protein-6 and Hepcidin Expression in Vivo *"

Article Title: The Role of Hepatocyte Hemojuvelin in the Regulation of Bone Morphogenic Protein-6 and Hepcidin Expression in Vivo *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.109488

Expression of Hjv in hepatocytes does not alter BMP6 expression. qRT-PCR analysis of BMP6 mRNA in the liver ( A ), BMP6 mRNA in the small intestine ( B ), and IL-6 mRNA in the liver ( C ) was performed using the cDNA preparations as described in the legend
Figure Legend Snippet: Expression of Hjv in hepatocytes does not alter BMP6 expression. qRT-PCR analysis of BMP6 mRNA in the liver ( A ), BMP6 mRNA in the small intestine ( B ), and IL-6 mRNA in the liver ( C ) was performed using the cDNA preparations as described in the legend

Techniques Used: Expressing, Quantitative RT-PCR

58) Product Images from "Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage"

Article Title: Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

Journal: BioMed Research International

doi: 10.1155/2015/708158

(a) TCP80 has increased binding to the p53 mRNA following DNA damage. MCF-7 cells were transfected with pcDNA3.1/HisB/TCP80 that encodes for the Xpress-tagged TCP80 protein. Twenty-four hours following transfection, the cells were treated with or without 10 μ M etoposide for 2 hours. They were then lysed in polysome lysis buffer and incubated with protein G-plus agarose beads coated with the anti-Xpress antibody. The TCP80 and mRNA complexes (mRNP) were immunoprecipitated and mRNA was extracted from the immunoprecipitate as described in Experimental procedures. RT-PCR was then performed to reverse-transcribe and amplify the p53 IRES sequence (~145 bp). (b) TCP80 positively affects the p53 IRES activity in response to DNA damage. MCF-7 cells were cotransfected with pRF or pR5UTRF along with either pcDNA3.1 or pcDNA3.1/HisB/TCP80. Twenty-four hours following the transfection, the cells were treated with or without etoposide for 2 hours. The cells were then lysed and a dual-luciferase assay was performed to detect firefly (Fluc) and renilla (Rluc) luciferase activities as described in Experimental procedures. The results presented are average ± SEM from three individual experiments.
Figure Legend Snippet: (a) TCP80 has increased binding to the p53 mRNA following DNA damage. MCF-7 cells were transfected with pcDNA3.1/HisB/TCP80 that encodes for the Xpress-tagged TCP80 protein. Twenty-four hours following transfection, the cells were treated with or without 10 μ M etoposide for 2 hours. They were then lysed in polysome lysis buffer and incubated with protein G-plus agarose beads coated with the anti-Xpress antibody. The TCP80 and mRNA complexes (mRNP) were immunoprecipitated and mRNA was extracted from the immunoprecipitate as described in Experimental procedures. RT-PCR was then performed to reverse-transcribe and amplify the p53 IRES sequence (~145 bp). (b) TCP80 positively affects the p53 IRES activity in response to DNA damage. MCF-7 cells were cotransfected with pRF or pR5UTRF along with either pcDNA3.1 or pcDNA3.1/HisB/TCP80. Twenty-four hours following the transfection, the cells were treated with or without etoposide for 2 hours. The cells were then lysed and a dual-luciferase assay was performed to detect firefly (Fluc) and renilla (Rluc) luciferase activities as described in Experimental procedures. The results presented are average ± SEM from three individual experiments.

Techniques Used: Binding Assay, Transfection, Lysis, Incubation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Activity Assay, Luciferase

59) Product Images from "Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study"

Article Title: Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

Journal: Virology Journal

doi: 10.1186/1743-422X-8-488

One-step assembly by homologous recombination in yeast of a ternary Yeast- Escherichia coli - Agrobacterium tumefaciens shuttle vector and simultaneous cloning of an ACLSV FL-cDNA to generate an infectious agroinoculable full-length viral cDNA clone . (A, B, C) Schematic representation of the strategy used to generate the four PCR fragments from which the construct is assembled by homologous recombination. The primers used are indicated by blue arrows near the respective PCR templates and are listed in Table 1. Regulatory elements are indicated by arrows of various colors. LB: T-DNA left border; 35S P: CaMV 35S promoter; 35S ter: CaMV 35S terminator; RB: T-DNA right border; ColE1: E. coli ColE1 origin of replication; oriV: A. tumefaciens oriV origin of replication; KanR: kanamycin resistance gene; 2μ Ori: yeast 2μ origin of replication; TRP1: yeast TRP1 selection gene. (A) Map of pBin61 from which are amplified the ~10.7 kbp fragment (PCR2, dark blue, carrying the 35S ter, the RB, the ColE1 and OriV replication signals and the KanR selection marker) and the ~2 kbp fragment (PCR4, yellow, carrying the LB and the 35S promoter) using respectively primer pairs ACPCR2F/PCR2R and PCR4F/ACPCR4R. (B) Map of Yeast-pBS70T used to amplify the ~2 kbp fragment (PCR3, red, carrying the yeast 2μ Ori-TRP1) using the PCR3F/PCR3R primer pair. (C) Genomic organization of ACLSV and position of the ACPCR1F/FLAC3 primer pair used to amplify the FL-viral cDNA (PCR1, light blue). (D) Non-denaturing 0.8% agarose gel electrophoresis of the four purified overlapping PCR products. (E) Schematic representation of the recombinant YEA-ACLSV ternary plasmid carrying the ACLSV FL-cDNA obtained by assembly by homologous recombination of the four fragments. The position of Eco RI sites used for restriction mapping of the plasmid is shown. (F) Non-denaturing 0.8% agarose gel electrophoresis of 5 independently obtained recombinant plasmids showing the expected Eco RI digestion pattern.
Figure Legend Snippet: One-step assembly by homologous recombination in yeast of a ternary Yeast- Escherichia coli - Agrobacterium tumefaciens shuttle vector and simultaneous cloning of an ACLSV FL-cDNA to generate an infectious agroinoculable full-length viral cDNA clone . (A, B, C) Schematic representation of the strategy used to generate the four PCR fragments from which the construct is assembled by homologous recombination. The primers used are indicated by blue arrows near the respective PCR templates and are listed in Table 1. Regulatory elements are indicated by arrows of various colors. LB: T-DNA left border; 35S P: CaMV 35S promoter; 35S ter: CaMV 35S terminator; RB: T-DNA right border; ColE1: E. coli ColE1 origin of replication; oriV: A. tumefaciens oriV origin of replication; KanR: kanamycin resistance gene; 2μ Ori: yeast 2μ origin of replication; TRP1: yeast TRP1 selection gene. (A) Map of pBin61 from which are amplified the ~10.7 kbp fragment (PCR2, dark blue, carrying the 35S ter, the RB, the ColE1 and OriV replication signals and the KanR selection marker) and the ~2 kbp fragment (PCR4, yellow, carrying the LB and the 35S promoter) using respectively primer pairs ACPCR2F/PCR2R and PCR4F/ACPCR4R. (B) Map of Yeast-pBS70T used to amplify the ~2 kbp fragment (PCR3, red, carrying the yeast 2μ Ori-TRP1) using the PCR3F/PCR3R primer pair. (C) Genomic organization of ACLSV and position of the ACPCR1F/FLAC3 primer pair used to amplify the FL-viral cDNA (PCR1, light blue). (D) Non-denaturing 0.8% agarose gel electrophoresis of the four purified overlapping PCR products. (E) Schematic representation of the recombinant YEA-ACLSV ternary plasmid carrying the ACLSV FL-cDNA obtained by assembly by homologous recombination of the four fragments. The position of Eco RI sites used for restriction mapping of the plasmid is shown. (F) Non-denaturing 0.8% agarose gel electrophoresis of 5 independently obtained recombinant plasmids showing the expected Eco RI digestion pattern.

Techniques Used: Homologous Recombination, Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Construct, Selection, Amplification, Marker, Agarose Gel Electrophoresis, Purification, Recombinant

60) Product Images from "Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis"

Article Title: Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis

Journal: Blood

doi: 10.1182/blood-2007-06-092338

Southern blot analysis of the murine kininogen gene. Genomic DNA from C57Bl/6J and 129J mice was digested with Eco RV and probed using a 32 P-dCTP–labeled PCR product spanning intron 4 and exon 4 of the kininogen gene. This probe hybridized to 2
Figure Legend Snippet: Southern blot analysis of the murine kininogen gene. Genomic DNA from C57Bl/6J and 129J mice was digested with Eco RV and probed using a 32 P-dCTP–labeled PCR product spanning intron 4 and exon 4 of the kininogen gene. This probe hybridized to 2

Techniques Used: Southern Blot, Mouse Assay, Labeling, Polymerase Chain Reaction

61) Product Images from "Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on Species D †"

Article Title: Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on Species D †

Journal: Journal of Virology

doi: 10.1128/JVI.00687-06

Tn-assisted cloning of the Ad19a genome. (A) Schematic representation of the Ad19a genome. The linear Ad genome is flanked by 135-bp ITRs (L-ITR, R-ITR; black and gray arrows). The HindIII fragments are marked according to size from A to G, with the HindIII B fragment shown in greater detail. Nonessential E3 ORFs are shown as black boxes and adjacent essential genes (100K, pVIII, fiber) as gray boxes. Numbers above or below the boxes indicate the names of the E3 ORFs based on their calculated molecular weights. (B) Schematic representation of Tn-assisted cloning of the Ad19a genome. The PCR-amplified Ad19a ITRs were cloned into the BAC vector pKSO carrying a chloramphenicol resistance gene (CmR), thereby generating pB19aLR with PacI (P) sites at each ITR-vector border. This entry vector was introduced into E. coli DH10B together with the recombination plasmid pBADαβγ expressing the respective λ genes involved in recombination. Purified Ad19a DNA was labeled in vitro with a Tn (white double arrows) carrying a kanamycin resistance (KnR) gene by use of TnsABC* transposase and the Tn donor plasmid pGPS1.1. Upon transformation with the Tn-labeled Ad19a DNA, ET recombination (ET) with p19aLR was induced and Ad19a-containing recombinant BACs (B19aTx) were selected by Kn and Cm. For simplicity, the HindIII site (H) present in the Tn is only shown in B19aTx. (C) HindIII digests of BAC DNA from selected Tn-positive clones (B19aT23, B19aT50, B19aT13, and B19aT51; lanes 1 to 4) and viral DNA from wt Ad19a and B19aT51-derived reconstituted virus, Ad19aT51 (lanes 5 and 6). The typical Ad19a HindIII fragments are indicated with the letters A to E (lane 5; F and G are not visible). Fragment A, one of the doublet DD′, and fragment E are visualized in all selected BAC clones. Fragment C and the other DD′ fragment are missing due to their linkage to the vector backbone. Together, these form fragment a. Insertion of the Tn in fragment B introduces an additional HindIII site, yielding two new fragments (marked by asterisks). For B19aT23 the second HindIII B-derived fragment is too small to be visible in this gel. Ad19aT51 was reconstituted by transfection of PacI-cleaved B19aT51 BAC DNA into 293 cells. PacI cleavage removes the plasmid vector; hence, viral DNA lacks fragment a and exhibits the normal end fragments (C and one of the DD′ fragments; compare lanes 5 and 6). Please note the presence of the same two extra fragments derived from fragment B (asterisk) in the recombinant Ad19aT51 virus as in the parental B19aT51 BAC (compare lanes 4 and 6).
Figure Legend Snippet: Tn-assisted cloning of the Ad19a genome. (A) Schematic representation of the Ad19a genome. The linear Ad genome is flanked by 135-bp ITRs (L-ITR, R-ITR; black and gray arrows). The HindIII fragments are marked according to size from A to G, with the HindIII B fragment shown in greater detail. Nonessential E3 ORFs are shown as black boxes and adjacent essential genes (100K, pVIII, fiber) as gray boxes. Numbers above or below the boxes indicate the names of the E3 ORFs based on their calculated molecular weights. (B) Schematic representation of Tn-assisted cloning of the Ad19a genome. The PCR-amplified Ad19a ITRs were cloned into the BAC vector pKSO carrying a chloramphenicol resistance gene (CmR), thereby generating pB19aLR with PacI (P) sites at each ITR-vector border. This entry vector was introduced into E. coli DH10B together with the recombination plasmid pBADαβγ expressing the respective λ genes involved in recombination. Purified Ad19a DNA was labeled in vitro with a Tn (white double arrows) carrying a kanamycin resistance (KnR) gene by use of TnsABC* transposase and the Tn donor plasmid pGPS1.1. Upon transformation with the Tn-labeled Ad19a DNA, ET recombination (ET) with p19aLR was induced and Ad19a-containing recombinant BACs (B19aTx) were selected by Kn and Cm. For simplicity, the HindIII site (H) present in the Tn is only shown in B19aTx. (C) HindIII digests of BAC DNA from selected Tn-positive clones (B19aT23, B19aT50, B19aT13, and B19aT51; lanes 1 to 4) and viral DNA from wt Ad19a and B19aT51-derived reconstituted virus, Ad19aT51 (lanes 5 and 6). The typical Ad19a HindIII fragments are indicated with the letters A to E (lane 5; F and G are not visible). Fragment A, one of the doublet DD′, and fragment E are visualized in all selected BAC clones. Fragment C and the other DD′ fragment are missing due to their linkage to the vector backbone. Together, these form fragment a. Insertion of the Tn in fragment B introduces an additional HindIII site, yielding two new fragments (marked by asterisks). For B19aT23 the second HindIII B-derived fragment is too small to be visible in this gel. Ad19aT51 was reconstituted by transfection of PacI-cleaved B19aT51 BAC DNA into 293 cells. PacI cleavage removes the plasmid vector; hence, viral DNA lacks fragment a and exhibits the normal end fragments (C and one of the DD′ fragments; compare lanes 5 and 6). Please note the presence of the same two extra fragments derived from fragment B (asterisk) in the recombinant Ad19aT51 virus as in the parental B19aT51 BAC (compare lanes 4 and 6).

Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, BAC Assay, Plasmid Preparation, Expressing, Purification, Labeling, In Vitro, Transformation Assay, Recombinant, Derivative Assay, Transfection

62) Product Images from "Next Generation Sequencing and Animal Models Reveal SLC9A3R1 as a New Gene Involved in Human Age-Related Hearing Loss"

Article Title: Next Generation Sequencing and Animal Models Reveal SLC9A3R1 as a New Gene Involved in Human Age-Related Hearing Loss

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2019.00142

CRISPR-Cas9 strategy for the generation of the R180Q- slc9a3r1 K/I model. (A) Comparison of human and zebrafish SLC9A3R1 amino acid sequence. Purple rectangle outlines R180 conservation among human and zebrafish. (B) Scheme displaying the nucleotide and amino acid translated sequence present at the target genome (below) and the ssOligo (above) used for modifying the target sequence. Below, green arrow in genome sequence outlines the sgRNA site. Above, red squares in ssOligo outline homologous recombination right and left arms. (C) Scheme displaying the PCR strategy used to identify potential mutants. (D) Example of R180Q-Slc9a3r1 mutant identified through the comparison of Sanger sequences obtained from a wild type (above) and a heterozygous individual (below). Both sequences display nucleotide and translated amino acid sequence. Above, targeted amino acid R180 is displayed in green. Below, modified amin oacid Q180 is displayed in red and modified nucleotides are displayed in red and lower case.
Figure Legend Snippet: CRISPR-Cas9 strategy for the generation of the R180Q- slc9a3r1 K/I model. (A) Comparison of human and zebrafish SLC9A3R1 amino acid sequence. Purple rectangle outlines R180 conservation among human and zebrafish. (B) Scheme displaying the nucleotide and amino acid translated sequence present at the target genome (below) and the ssOligo (above) used for modifying the target sequence. Below, green arrow in genome sequence outlines the sgRNA site. Above, red squares in ssOligo outline homologous recombination right and left arms. (C) Scheme displaying the PCR strategy used to identify potential mutants. (D) Example of R180Q-Slc9a3r1 mutant identified through the comparison of Sanger sequences obtained from a wild type (above) and a heterozygous individual (below). Both sequences display nucleotide and translated amino acid sequence. Above, targeted amino acid R180 is displayed in green. Below, modified amin oacid Q180 is displayed in red and modified nucleotides are displayed in red and lower case.

Techniques Used: CRISPR, Sequencing, Homologous Recombination, Polymerase Chain Reaction, Mutagenesis, Modification

63) Product Images from "Developmental lung expression and transcriptional regulation of Claudin-6 by TTF-1, Gata-6, and FoxA2"

Article Title: Developmental lung expression and transcriptional regulation of Claudin-6 by TTF-1, Gata-6, and FoxA2

Journal: Respiratory Research

doi: 10.1186/1465-9921-15-70

Cldn6 mRNA Expression . Control C57Bl6 mice were screened by microarray analysis and Cldn6 expression levels were derived relative to GAPDH from E15-PN0 (A) . Confirmatory quantitative RT-PCR was conducted using total RNA from embryonic C57Bl6 mice and results are presented relative to GAPDH (B) . Representative data from experiments performed in triplicate are shown. *P ≤ 0.05 when comparisons were made between E15.5 and E16.5 or E15.5 and E17.5.
Figure Legend Snippet: Cldn6 mRNA Expression . Control C57Bl6 mice were screened by microarray analysis and Cldn6 expression levels were derived relative to GAPDH from E15-PN0 (A) . Confirmatory quantitative RT-PCR was conducted using total RNA from embryonic C57Bl6 mice and results are presented relative to GAPDH (B) . Representative data from experiments performed in triplicate are shown. *P ≤ 0.05 when comparisons were made between E15.5 and E16.5 or E15.5 and E17.5.

Techniques Used: Expressing, Mouse Assay, Microarray, Derivative Assay, Quantitative RT-PCR

64) Product Images from "Blockade of HIV-1 Infection of New World Monkey Cells Occurs Primarily at the Stage of Virus Entry"

Article Title: Blockade of HIV-1 Infection of New World Monkey Cells Occurs Primarily at the Stage of Virus Entry

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20020468

Predicted amino acid alignment of Ig domains 1 and 2 of New World monkey CD4 proteins. PCR primers specific for the 5′ and 3′ untranslated regions were used to amplify CD4 from cDNA generated from squirrel monkey or marmoset PBMCs. (A) The predicted amino acid sequences of these cloned CD4 molecules are aligned to the previously identified sequences of human, rhesus macaque, and murine CD4. The shaded boxes designate Ig domains 1 and 2. The solid line identifies the signal sequence, which is cleaved from the mature protein. Dots (.) denote residues identical to the human sequence and minus signs (−) denote deletions. Gray arrows denote residues that differ from the published squirrel monkey CD4 allele D86588 . GenBank/EMBL/DDBJ accession nos. for the sequences are as follows: human, M12807 ; rhesus, D63347 ; squirrel monkey, AF452617 ; common marmoset, AF452616 ; mouse, NM_013488. (B) The region of human CD4 that makes important contacts with the HIV-1 gp120 glycoprotein is aligned with the analogous region of squirrel monkey and common marmoset CD4 proteins. Phenylalanine 43 and arginine 59 (arrows) are particularly important residues for gp120 binding (references 45, 77, and 79). Changes introduced into the squirrel monkey and common marmoset CD4 molecules in this study are shown in bold.
Figure Legend Snippet: Predicted amino acid alignment of Ig domains 1 and 2 of New World monkey CD4 proteins. PCR primers specific for the 5′ and 3′ untranslated regions were used to amplify CD4 from cDNA generated from squirrel monkey or marmoset PBMCs. (A) The predicted amino acid sequences of these cloned CD4 molecules are aligned to the previously identified sequences of human, rhesus macaque, and murine CD4. The shaded boxes designate Ig domains 1 and 2. The solid line identifies the signal sequence, which is cleaved from the mature protein. Dots (.) denote residues identical to the human sequence and minus signs (−) denote deletions. Gray arrows denote residues that differ from the published squirrel monkey CD4 allele D86588 . GenBank/EMBL/DDBJ accession nos. for the sequences are as follows: human, M12807 ; rhesus, D63347 ; squirrel monkey, AF452617 ; common marmoset, AF452616 ; mouse, NM_013488. (B) The region of human CD4 that makes important contacts with the HIV-1 gp120 glycoprotein is aligned with the analogous region of squirrel monkey and common marmoset CD4 proteins. Phenylalanine 43 and arginine 59 (arrows) are particularly important residues for gp120 binding (references 45, 77, and 79). Changes introduced into the squirrel monkey and common marmoset CD4 molecules in this study are shown in bold.

Techniques Used: Polymerase Chain Reaction, Generated, Clone Assay, Sequencing, Binding Assay

65) Product Images from "Molecular Engineering of High Affinity Single-chain Antibody Fragment for Endothelial Targeting of Proteins and Nanocarriers in Rodents and Humans"

Article Title: Molecular Engineering of High Affinity Single-chain Antibody Fragment for Endothelial Targeting of Proteins and Nanocarriers in Rodents and Humans

Journal: Journal of controlled release : official journal of the Controlled Release Society

doi: 10.1016/j.jconrel.2016.02.006

PCR amplication of Ab62 V H and V L cDNAs. (a) Schematic comparing use of traditional (FR1) vs. signal peptide (SP) primer sets. SP primers amplify full-length cDNA, without incorporation of primer-derived sequence, which can introduce amino acid substitutions at the N terminus of the FR1 region. (b) DNA gels comparing V L cDNAs amplified using FR1 vs. SP primer sets. Sequencing of each appropriately sized amplicon revealed an additional advantage of the SP primers – selective amplification of the Ab62 anti-PECAM V L by primers MKV.SP6 and SP9.
Figure Legend Snippet: PCR amplication of Ab62 V H and V L cDNAs. (a) Schematic comparing use of traditional (FR1) vs. signal peptide (SP) primer sets. SP primers amplify full-length cDNA, without incorporation of primer-derived sequence, which can introduce amino acid substitutions at the N terminus of the FR1 region. (b) DNA gels comparing V L cDNAs amplified using FR1 vs. SP primer sets. Sequencing of each appropriately sized amplicon revealed an additional advantage of the SP primers – selective amplification of the Ab62 anti-PECAM V L by primers MKV.SP6 and SP9.

Techniques Used: Polymerase Chain Reaction, Derivative Assay, Sequencing, Introduce, Amplification

66) Product Images from "Cooperation between Viral Interferon Regulatory Factor 4 and RTA To Activate a Subset of Kaposi's Sarcoma-Associated Herpesvirus Lytic Promoters"

Article Title: Cooperation between Viral Interferon Regulatory Factor 4 and RTA To Activate a Subset of Kaposi's Sarcoma-Associated Herpesvirus Lytic Promoters

Journal: Journal of Virology

doi: 10.1128/JVI.00694-11

vIRF4 contributes to KSHV reactivation. (A) KSHV latently infected iSLK.219 cells were either mock treated or transfected with empty vector or vector expressing full-length vIRF4. The next day, the transfected cultures were induced with 100 ng/ml doxycycline (DOX) to drive expression of a DOX-regulated RTA cDNA stably integrated into the cell genome and maintained for 72 h. Culture medium was collected, filtered to remove debris, including cells, and used to infect 293-PAN-Luc reporter cells. After 48 h, lysates were prepared and assayed for luciferase activity. Values represent the means and standard errors of the means of three independent transfections. (B) iSLK.219 cells were transfected with dsiRNAs against EGFP or K10/vIRF4 mRNA sequences. Cultures were induced to reactivate by using doxycycline, and RNA was harvested 72 h later and analyzed by quantitative RT-PCR using primers to detect K9/vIRF1 (open bars) and K10/vIRF4 (filled bars). (C) Results of an experiment similar to that shown in panel B, except that the culture medium was collected from induced and uninduced iSLK.219 cells and assayed for infectious KSHV virions by using 293-PAN-Luc cells. Values are expressed relative to the averages of the uninduced samples.
Figure Legend Snippet: vIRF4 contributes to KSHV reactivation. (A) KSHV latently infected iSLK.219 cells were either mock treated or transfected with empty vector or vector expressing full-length vIRF4. The next day, the transfected cultures were induced with 100 ng/ml doxycycline (DOX) to drive expression of a DOX-regulated RTA cDNA stably integrated into the cell genome and maintained for 72 h. Culture medium was collected, filtered to remove debris, including cells, and used to infect 293-PAN-Luc reporter cells. After 48 h, lysates were prepared and assayed for luciferase activity. Values represent the means and standard errors of the means of three independent transfections. (B) iSLK.219 cells were transfected with dsiRNAs against EGFP or K10/vIRF4 mRNA sequences. Cultures were induced to reactivate by using doxycycline, and RNA was harvested 72 h later and analyzed by quantitative RT-PCR using primers to detect K9/vIRF1 (open bars) and K10/vIRF4 (filled bars). (C) Results of an experiment similar to that shown in panel B, except that the culture medium was collected from induced and uninduced iSLK.219 cells and assayed for infectious KSHV virions by using 293-PAN-Luc cells. Values are expressed relative to the averages of the uninduced samples.

Techniques Used: Infection, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Luciferase, Activity Assay, Quantitative RT-PCR

67) Product Images from "Mannose receptor regulates myoblast motility and muscle growth"

Article Title: Mannose receptor regulates myoblast motility and muscle growth

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200601102

MR is expressed in muscle cells during myoblast fusion. (A) Primary myoblasts (Mb) were induced to differentiate for 24 or 48 h. MR mRNA was analyzed by RT-PCR. Myogenin mRNA was assessed as a marker of myogenic differentiation. Phase-contrast images of muscle cells are shown to illustrate fusion progress at each time point. MR, 390 bp; Myogenin, 266 bp; 18S, 488 bp. (B) Representative images of muscle cells after 24 h of differentiation immunostained with an antibody against MR. Bar, 50 μm. (C) Primary myoblasts were differentiated for 24 h and subsequently treated with vehicle or 10 ng/ml IL-4 for 24 h. MR mRNA was analyzed by RT-PCR. (D) MR mRNA expression in WT or IL-4 receptor α-null (IL-4Rα −/− ) myotubes after 48 h in DM was examined by RT-PCR. MR, 390 bp; 18S, 488 bp. Representative ethidium bromide staining of agarose gels is shown with 18S ribosomal RNA as an internal control for all RT-PCR analyses. All data are indicative of results from three independent cell isolates.
Figure Legend Snippet: MR is expressed in muscle cells during myoblast fusion. (A) Primary myoblasts (Mb) were induced to differentiate for 24 or 48 h. MR mRNA was analyzed by RT-PCR. Myogenin mRNA was assessed as a marker of myogenic differentiation. Phase-contrast images of muscle cells are shown to illustrate fusion progress at each time point. MR, 390 bp; Myogenin, 266 bp; 18S, 488 bp. (B) Representative images of muscle cells after 24 h of differentiation immunostained with an antibody against MR. Bar, 50 μm. (C) Primary myoblasts were differentiated for 24 h and subsequently treated with vehicle or 10 ng/ml IL-4 for 24 h. MR mRNA was analyzed by RT-PCR. (D) MR mRNA expression in WT or IL-4 receptor α-null (IL-4Rα −/− ) myotubes after 48 h in DM was examined by RT-PCR. MR, 390 bp; 18S, 488 bp. Representative ethidium bromide staining of agarose gels is shown with 18S ribosomal RNA as an internal control for all RT-PCR analyses. All data are indicative of results from three independent cell isolates.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Marker, Expressing, Staining

68) Product Images from "Human cytomegalovirus miR-UL112-1 promotes the down-regulation of viral immediate early-gene expression during latency to prevent T-cell recognition of latently infected cells"

Article Title: Human cytomegalovirus miR-UL112-1 promotes the down-regulation of viral immediate early-gene expression during latency to prevent T-cell recognition of latently infected cells

Journal: The Journal of General Virology

doi: 10.1099/jgv.0.000546

Latent infection with Δ112-1 target site mutant virus results in increased levels of IE72 RNA expression compared with WT virus. THP-1 cells were infected at an m.o.i. of 5 with the parental WT virus or the Δ112-1 target site mutant. Latently infected cells were sorted for GFP expression at 2 days post-infection, before mRNA expression was analysed by quantitative reverse transcriptase PCR at 3 days post-infection. The level of UL138 and IE72 normalized to the mRNA level of housekeeping gene GAPDH is shown in (a) and (b), respectively. Data shown is representative of three independent repeats, whilst error bars shown mark standard deviation of technical replicates.
Figure Legend Snippet: Latent infection with Δ112-1 target site mutant virus results in increased levels of IE72 RNA expression compared with WT virus. THP-1 cells were infected at an m.o.i. of 5 with the parental WT virus or the Δ112-1 target site mutant. Latently infected cells were sorted for GFP expression at 2 days post-infection, before mRNA expression was analysed by quantitative reverse transcriptase PCR at 3 days post-infection. The level of UL138 and IE72 normalized to the mRNA level of housekeeping gene GAPDH is shown in (a) and (b), respectively. Data shown is representative of three independent repeats, whilst error bars shown mark standard deviation of technical replicates.

Techniques Used: Infection, Mutagenesis, RNA Expression, Expressing, Polymerase Chain Reaction, Standard Deviation

69) Product Images from "The Chlamydia trachomatis Plasmid Is a Transcriptional Regulator of Chromosomal Genes and a Virulence Factor "

Article Title: The Chlamydia trachomatis Plasmid Is a Transcriptional Regulator of Chromosomal Genes and a Virulence Factor

Journal: Infection and Immunity

doi: 10.1128/IAI.00102-08

qRT-PCR of glycogen metabolic genes shows a significant difference in glgA expression between L2(434) and L2(25667R). McCoy cells were infected with L2(434) or L2(25667R) (MOI of 1.0) and harvested for RNA and DNA at various times p.i. (PI) (A) Genome
Figure Legend Snippet: qRT-PCR of glycogen metabolic genes shows a significant difference in glgA expression between L2(434) and L2(25667R). McCoy cells were infected with L2(434) or L2(25667R) (MOI of 1.0) and harvested for RNA and DNA at various times p.i. (PI) (A) Genome

Techniques Used: Quantitative RT-PCR, Expressing, Infection

70) Product Images from "Thyroid hormone-regulated gene expression in juvenile mouse liver: identification of thyroid response elements using microarray profiling and in silico analyses"

Article Title: Thyroid hormone-regulated gene expression in juvenile mouse liver: identification of thyroid response elements using microarray profiling and in silico analyses

Journal: BMC Genomics

doi: 10.1186/1471-2164-12-634

Relative enrichment of newly identified TREs determined by ChIP-PCR . The top half of each section shows the amplicons run on an agarose gel, and the bottom half shows relative enrichment of the immunoprecipitated (IP) samples and the total input (TI) samples when compared to β-actin enrichment. ChIP-PCR validation of negative (β-actin) and positive (Mlxipl) controls are presented in section (A). Sections (B) to (E) show enrichment of Tor1a, H01Rik (2310003H01Rik), Hectd3 and Slc25a45. Asterisks (*) denote a significant difference, p ≤ 0.05, determined by student t-test. The mean enrichments (± standard deviations) are also presented for each section. All presented immunoprecipitated enrichments were significant (p ≤ 0.05 determined by student t-test) when compared to total input, except for β-actin.
Figure Legend Snippet: Relative enrichment of newly identified TREs determined by ChIP-PCR . The top half of each section shows the amplicons run on an agarose gel, and the bottom half shows relative enrichment of the immunoprecipitated (IP) samples and the total input (TI) samples when compared to β-actin enrichment. ChIP-PCR validation of negative (β-actin) and positive (Mlxipl) controls are presented in section (A). Sections (B) to (E) show enrichment of Tor1a, H01Rik (2310003H01Rik), Hectd3 and Slc25a45. Asterisks (*) denote a significant difference, p ≤ 0.05, determined by student t-test. The mean enrichments (± standard deviations) are also presented for each section. All presented immunoprecipitated enrichments were significant (p ≤ 0.05 determined by student t-test) when compared to total input, except for β-actin.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Immunoprecipitation

71) Product Images from "The CD83 reporter mouse elucidates the activity of the CD83 promoter in B, T, and dendritic cell populations in vivo"

Article Title: The CD83 reporter mouse elucidates the activity of the CD83 promoter in B, T, and dendritic cell populations in vivo

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0806335105

Generation of CD83-IRES2-EGFP reporter mice. ( A ) CD83 knockin gene targeting strategy. Untranslated regions, filled boxes; ORFs, open boxes; PCR primers, arrowheads; DT-A, diphtheria toxin subunit A. The Southern blot probes a, b, and neo are indicated by underlining. ( B ) Southern blot analysis of BamHI-digested DNA from ES cell clones. het, heterozygous; wt, wild type; wt band, filled arrow; mutant band, open arrow. ( C ) PCR screening of tail DNA of four littermate progeny of CD83-IRES2-EGFP +/− mice backcrossed to C57Bl6 (Bl6) mice. Control, water.
Figure Legend Snippet: Generation of CD83-IRES2-EGFP reporter mice. ( A ) CD83 knockin gene targeting strategy. Untranslated regions, filled boxes; ORFs, open boxes; PCR primers, arrowheads; DT-A, diphtheria toxin subunit A. The Southern blot probes a, b, and neo are indicated by underlining. ( B ) Southern blot analysis of BamHI-digested DNA from ES cell clones. het, heterozygous; wt, wild type; wt band, filled arrow; mutant band, open arrow. ( C ) PCR screening of tail DNA of four littermate progeny of CD83-IRES2-EGFP +/− mice backcrossed to C57Bl6 (Bl6) mice. Control, water.

Techniques Used: Mouse Assay, Knock-In, Polymerase Chain Reaction, Southern Blot, Clone Assay, Mutagenesis

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Article Snippet: .. In selected cases, the full-length WHV DNA was amplified with primers PPC1 (1908-1926) and XPC (1891-1907) located at the WHV nick region, using the Expand High Fidelity PCR System (Roche Diagnostics, Laval, Quebec, Canada) and conditions described previously ( ). .. When required, complete WHV DNA was further amplified with WHV C (pair PCNV-COR) and S (pair PSW-SUW) gene–specific primers.

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: .. HBP1-MYC constructs Truncated Hbp1 cDNA constructs were amplified from 12.5 dpc testis cDNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) with the common forward primer All-Hbp1-MYC-F containing a BamHI restriction site and individual reverse primers containing an XhoI restriction site, FlHbp1-MYC-R and ΔHbp1-MYC-R ( ). .. These fragments were cloned into a modified pcDNA3.0 vector generating an N-terminal MYC-tagged protein and sequenced for verification.

Article Title: Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells
Article Snippet: Cadherin-11 3′-UTR fragment was cloned from genomic DNA obtained from MDA-MB-231 cells using Expand High Fidelity PCR System (11732641001, Roche, Indianapolis, IN) according to the manufacturer's protocol. .. For amplification of the CDH11 3′UTR NCBI forward: ( 5′-TGCTAGCTAAGTAAGTAACAATAACGATACAAATTT-3′ ) and reverse: ( 5′-CCGGATCCACGCGTGAATCTTGTCTGAAAAAACATTTG-3′ ) primers were used.

Article Title: Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis
Article Snippet: .. Promoter cloning The DNA fragment (3.1 kb), containing the MLK3 promoter was amplified from the HEK293 genomic DNA using the Expand High Fidelity PCR system (Roche Applied Science) and designated MLK3-P1. .. Five different deletion fragments (Figure ) were also generated using MLK3-P1 as a template and sub-cloned into pGL4.16 [luc2CP/Hygro] vector (Promega).

Article Title: The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A
Article Snippet: PCR was performed with the oligonucleotide designed to amplify the linear pMT2A-Luc altered the imperfect XRE2: Fwd, 5′-CACGaaaccGGCACCCAGCACC-3′ and Rev, 5′CGCCTCCGCCGTGTGCACAG-3′ using Expand High Fidelity PCR System (Roche Applied Science, Mannheim, Germany). .. Human AHR cDNA was amplified from pBluescriptR-human AHR (MHS1010–98075336, Open Biosystems) using the primer: Fwd, 5′-CTCGAGGGATGAACAGCAGCAGCGCC-3′ and Rev, 5′-CTCGAGTTACAGGAATCCACTGGATGTCAA-3′.

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: .. Generation of the 2KbHbp1P_pHSP68_LacZ mouse lines The 2 kb Hbp1 proximal promoter region was amplified from C57Bl/6 genomic DNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) using primers Hbp1P2kb-F and Hbp1P2kb-R ( ). .. The region included -2121 bp to +1 bp relative to the Hbp1 transcription start site and was cloned into a modified pBluescript vector containing LacZ driven by the minimal HSP68 promoter.

Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine
Article Snippet: .. Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI). .. The forward primer contained a XhoI restriction site, and the reverse primer contained a SacI restriction site for cloning into the XhoI/SacI sites in the multiple cloning site of the pBigT plasmid to create pBigT-Gata4 ( A ).

Article Title: Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment
Article Snippet: .. Constructs The mouse and human Use1 UTR was amplified using the Expand High Fidelity PCR System (Roche), a forward primer harboring an XhoI site and T7 promoter, and a reverse primer containing a NcoI site. .. PCR products were ligated in pCR2.1 (Invitrogen) and pGL2-basic.

Article Title: Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat
Article Snippet: .. Gene fragments for insertion into BSMV were PCR amplified using Expand High Fidelity PCR System (Roche Diagnostics, Mannheim, Germany) with the primers shown in Additional file . ..

Article Title: Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues
Article Snippet: .. Generation and analysis of dronc reporter lacZ lines 1.64 kb and 1.33 kb of the dronc promoter were PCR amplified from Drosophila genomic DNA using Expand High Fidelity PCR System (Roche) with the primer sets DrPrF3cBglII (5′ CCG AGA TCT ATG TAC GTT ATG TTA TAG TAA GTG TA 3′); DrPrR1BglII (5′ CGG AGA TCT CCG GAT ATG GCT TCC ACG CGT 3′) and DrPrF3eBglII (5′ CGA AGA TCT AAT TGT GTA CAA CTA AAG GAA 3′); DrPrR1BglII, respectively. .. PCR products were cloned into pGem-T easy (Promega), and then subcloned into the BglII site of the p-element transformation lacZ reporter vector pCaSpeR-NLSlacZ (provided by Carl Thummel, Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT) after BglII digestion.

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: .. The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche). .. The following primer pairs were used to amplify the homology arms: 5′-gtc gac aga agt tct cgg agc ggg tgc tga act-3′, 5′-atc ctc cat gac agt tat ctg ca-3′ (for 5′ homology arm); 5′-tag gat cca caa agt cac aga gcc act ttc a-3′, 5′-tag gat ccc ctc tga ctt att ctt gct ct tag-3′ (for 3′ homology arm).

Article Title: HIV Restriction by APOBEC3 in Humanized Mice
Article Snippet: .. Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche). .. All PCR primers amplify both HIV-1JR-CSF and HIV-1LAI and were designed to anneal in regions with the fewest possible putative APOBEC3 deamination sites to avoid potential primer mismatch due to APOBEC3 induced mutagenesis.

Reporter Assay:

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: We compared the activity of PGK and CAG promoters in rat ES cells using the Dual-Luciferase Reporter Assay (Promega). .. The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche).

Synthesized:

Article Title: Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat
Article Snippet: VIGS constructs cDNA was synthesized from 1 μg of total RNA using oligo(dT)15 primer (Promega, Madison, WI, USA) and M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). .. Gene fragments for insertion into BSMV were PCR amplified using Expand High Fidelity PCR System (Roche Diagnostics, Mannheim, Germany) with the primers shown in Additional file .

Construct:

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: .. HBP1-MYC constructs Truncated Hbp1 cDNA constructs were amplified from 12.5 dpc testis cDNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) with the common forward primer All-Hbp1-MYC-F containing a BamHI restriction site and individual reverse primers containing an XhoI restriction site, FlHbp1-MYC-R and ΔHbp1-MYC-R ( ). .. These fragments were cloned into a modified pcDNA3.0 vector generating an N-terminal MYC-tagged protein and sequenced for verification.

Article Title: Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells
Article Snippet: Dominant negative (DN) Snail was generated by PCR from the wild type Snail construct using primers described in Yamasaki et al. forward: ( 5′-CGGGATCCACTATGGCCTTCAACTGCAAATACTG-3′ ) and reverse: ( 5′-CGCTCGAGGCGGGGACATCCTGAGCA-3′ ) and cloned into pCMV-Tag2 (Stratagene, La Jolla, CA) using BamHI and XhoI restriction sites . .. Cadherin-11 3′-UTR fragment was cloned from genomic DNA obtained from MDA-MB-231 cells using Expand High Fidelity PCR System (11732641001, Roche, Indianapolis, IN) according to the manufacturer's protocol.

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells
Article Snippet: The targeting construct was designed to introduce loxP sites flanking exons 2 and 3, which encode part of the DNA-binding domain. .. The flanks and “deletable” fragment of the Ebf1 gene were PCR-cloned with the Expand High-Fidelity PCR system (Roche) using genomic DNA from D3V embryonic stem cells as a template.

Article Title: Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment
Article Snippet: .. Constructs The mouse and human Use1 UTR was amplified using the Expand High Fidelity PCR System (Roche), a forward primer harboring an XhoI site and T7 promoter, and a reverse primer containing a NcoI site. .. PCR products were ligated in pCR2.1 (Invitrogen) and pGL2-basic.

Article Title: Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat
Article Snippet: Paragraph title: VIGS constructs ... Gene fragments for insertion into BSMV were PCR amplified using Expand High Fidelity PCR System (Roche Diagnostics, Mannheim, Germany) with the primers shown in Additional file .

Article Title: Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues
Article Snippet: Generation and analysis of dronc reporter lacZ lines 1.64 kb and 1.33 kb of the dronc promoter were PCR amplified from Drosophila genomic DNA using Expand High Fidelity PCR System (Roche) with the primer sets DrPrF3cBglII (5′ CCG AGA TCT ATG TAC GTT ATG TTA TAG TAA GTG TA 3′); DrPrR1BglII (5′ CGG AGA TCT CCG GAT ATG GCT TCC ACG CGT 3′) and DrPrF3eBglII (5′ CGA AGA TCT AAT TGT GTA CAA CTA AAG GAA 3′); DrPrR1BglII, respectively. .. The 2.8-kb dronc -LacZ reporter construct has been described previously ( ).

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: We then constructed a p53 gene targeting vector using the CAG promoter to drive the expression of the EGFP-IRES-Pac selection cassette. .. The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche).

Modification:

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: HBP1-MYC constructs Truncated Hbp1 cDNA constructs were amplified from 12.5 dpc testis cDNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) with the common forward primer All-Hbp1-MYC-F containing a BamHI restriction site and individual reverse primers containing an XhoI restriction site, FlHbp1-MYC-R and ΔHbp1-MYC-R ( ). .. These fragments were cloned into a modified pcDNA3.0 vector generating an N-terminal MYC-tagged protein and sequenced for verification.

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: Generation of the 2KbHbp1P_pHSP68_LacZ mouse lines The 2 kb Hbp1 proximal promoter region was amplified from C57Bl/6 genomic DNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) using primers Hbp1P2kb-F and Hbp1P2kb-R ( ). .. The region included -2121 bp to +1 bp relative to the Hbp1 transcription start site and was cloned into a modified pBluescript vector containing LacZ driven by the minimal HSP68 promoter.

Activity Assay:

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: The activity of the CAG promoter was 36 times higher than that of the PGK promoter. .. The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche).

Infection:

Article Title: HIV Restriction by APOBEC3 in Humanized Mice
Article Snippet: Paragraph title: Molecular analysis of HIV-1 infection ... Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche).

Expressing:

Article Title: Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells
Article Snippet: Paragraph title: Expression Vectors ... Cadherin-11 3′-UTR fragment was cloned from genomic DNA obtained from MDA-MB-231 cells using Expand High Fidelity PCR System (11732641001, Roche, Indianapolis, IN) according to the manufacturer's protocol.

Article Title: The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A
Article Snippet: PCR was performed with the oligonucleotide designed to amplify the linear pMT2A-Luc altered the imperfect XRE2: Fwd, 5′-CACGaaaccGGCACCCAGCACC-3′ and Rev, 5′CGCCTCCGCCGTGTGCACAG-3′ using Expand High Fidelity PCR System (Roche Applied Science, Mannheim, Germany). .. This linear PCR product was phosphorylated and ligated itself after digestion of the template plasmid with Dpn I. AHR and GR expression plasmids were prepared as follows.

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: We then constructed a p53 gene targeting vector using the CAG promoter to drive the expression of the EGFP-IRES-Pac selection cassette. .. The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche).

Knock-In:

Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine
Article Snippet: .. Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI). .. The forward primer contained a XhoI restriction site, and the reverse primer contained a SacI restriction site for cloning into the XhoI/SacI sites in the multiple cloning site of the pBigT plasmid to create pBigT-Gata4 ( A ).

Transformation Assay:

Article Title: Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues
Article Snippet: Generation and analysis of dronc reporter lacZ lines 1.64 kb and 1.33 kb of the dronc promoter were PCR amplified from Drosophila genomic DNA using Expand High Fidelity PCR System (Roche) with the primer sets DrPrF3cBglII (5′ CCG AGA TCT ATG TAC GTT ATG TTA TAG TAA GTG TA 3′); DrPrR1BglII (5′ CGG AGA TCT CCG GAT ATG GCT TCC ACG CGT 3′) and DrPrF3eBglII (5′ CGA AGA TCT AAT TGT GTA CAA CTA AAG GAA 3′); DrPrR1BglII, respectively. .. PCR products were cloned into pGem-T easy (Promega), and then subcloned into the BglII site of the p-element transformation lacZ reporter vector pCaSpeR-NLSlacZ (provided by Carl Thummel, Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT) after BglII digestion.

Derivative Assay:

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells
Article Snippet: The flanks and “deletable” fragment of the Ebf1 gene were PCR-cloned with the Expand High-Fidelity PCR system (Roche) using genomic DNA from D3V embryonic stem cells as a template. .. Chimeras were obtained after injection of the targeted embryonic D3V stem cell clones into blastocysts derived from C57BL/6 mice.

Countercurrent Chromatography:

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche). .. The following primer pairs were used to amplify the homology arms: 5′-gtc gac aga agt tct cgg agc ggg tgc tga act-3′, 5′-atc ctc cat gac agt tat ctg ca-3′ (for 5′ homology arm); 5′-tag gat cca caa agt cac aga gcc act ttc a-3′, 5′-tag gat ccc ctc tga ctt att ctt gct ct tag-3′ (for 3′ homology arm).

Introduce:

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells
Article Snippet: The targeting construct was designed to introduce loxP sites flanking exons 2 and 3, which encode part of the DNA-binding domain. .. The flanks and “deletable” fragment of the Ebf1 gene were PCR-cloned with the Expand High-Fidelity PCR system (Roche) using genomic DNA from D3V embryonic stem cells as a template.

Multiple Displacement Amplification:

Article Title: Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells
Article Snippet: .. Cadherin-11 3′-UTR fragment was cloned from genomic DNA obtained from MDA-MB-231 cells using Expand High Fidelity PCR System (11732641001, Roche, Indianapolis, IN) according to the manufacturer's protocol. .. For amplification of the CDH11 3′UTR NCBI forward: ( 5′-TGCTAGCTAAGTAAGTAACAATAACGATACAAATTT-3′ ) and reverse: ( 5′-CCGGATCCACGCGTGAATCTTGTCTGAAAAAACATTTG-3′ ) primers were used.

Generated:

Article Title: Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells
Article Snippet: Dominant negative (DN) Snail was generated by PCR from the wild type Snail construct using primers described in Yamasaki et al. forward: ( 5′-CGGGATCCACTATGGCCTTCAACTGCAAATACTG-3′ ) and reverse: ( 5′-CGCTCGAGGCGGGGACATCCTGAGCA-3′ ) and cloned into pCMV-Tag2 (Stratagene, La Jolla, CA) using BamHI and XhoI restriction sites . .. Cadherin-11 3′-UTR fragment was cloned from genomic DNA obtained from MDA-MB-231 cells using Expand High Fidelity PCR System (11732641001, Roche, Indianapolis, IN) according to the manufacturer's protocol.

Article Title: Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis
Article Snippet: Promoter cloning The DNA fragment (3.1 kb), containing the MLK3 promoter was amplified from the HEK293 genomic DNA using the Expand High Fidelity PCR system (Roche Applied Science) and designated MLK3-P1. .. Five different deletion fragments (Figure ) were also generated using MLK3-P1 as a template and sub-cloned into pGL4.16 [luc2CP/Hygro] vector (Promega).

Article Title: Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment
Article Snippet: Constructs The mouse and human Use1 UTR was amplified using the Expand High Fidelity PCR System (Roche), a forward primer harboring an XhoI site and T7 promoter, and a reverse primer containing a NcoI site. .. The plasmids ΔSS, Δ GGGG and Δ AGGGCGGA were generated with the QuickChange Site-Direct Mutagenesis Kit (Stratagene).

Article Title: HIV Restriction by APOBEC3 in Humanized Mice
Article Snippet: Viral RNA was isolated from plasma using QIAamp viral RNA columns (Qiagen) according to the manufacture's protocol including an optional treatment with RNase-free DNase (Qiagen) during extraction and cDNA was generated using Superscript III Reverse Transcriptase (Invitrogen). .. Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche).

Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors
Article Snippet: We generated fluorescence-labeled probes (Fluorescein) for the gfp, msp2, SSUD and SSU-Api genes using the Label IT Nucleic Acid Labeling kit according to the instructions of the manufacturer (Mirus, MIR 3225). .. Briefly, probes were made by PCR from 3D7 WT gDNA for gfp, msp2, SSUD and SSU-Api using Expand High Fidelity PCR System (Roche, 3300226) with 0.3 M primers in a 50 μl reaction.

Polymerase Chain Reaction:

Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis
Article Snippet: .. In selected cases, the full-length WHV DNA was amplified with primers PPC1 (1908-1926) and XPC (1891-1907) located at the WHV nick region, using the Expand High Fidelity PCR System (Roche Diagnostics, Laval, Quebec, Canada) and conditions described previously ( ). .. When required, complete WHV DNA was further amplified with WHV C (pair PCNV-COR) and S (pair PSW-SUW) gene–specific primers.

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: .. HBP1-MYC constructs Truncated Hbp1 cDNA constructs were amplified from 12.5 dpc testis cDNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) with the common forward primer All-Hbp1-MYC-F containing a BamHI restriction site and individual reverse primers containing an XhoI restriction site, FlHbp1-MYC-R and ΔHbp1-MYC-R ( ). .. These fragments were cloned into a modified pcDNA3.0 vector generating an N-terminal MYC-tagged protein and sequenced for verification.

Article Title: Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells
Article Snippet: .. Cadherin-11 3′-UTR fragment was cloned from genomic DNA obtained from MDA-MB-231 cells using Expand High Fidelity PCR System (11732641001, Roche, Indianapolis, IN) according to the manufacturer's protocol. .. For amplification of the CDH11 3′UTR NCBI forward: ( 5′-TGCTAGCTAAGTAAGTAACAATAACGATACAAATTT-3′ ) and reverse: ( 5′-CCGGATCCACGCGTGAATCTTGTCTGAAAAAACATTTG-3′ ) primers were used.

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells
Article Snippet: .. The flanks and “deletable” fragment of the Ebf1 gene were PCR-cloned with the Expand High-Fidelity PCR system (Roche) using genomic DNA from D3V embryonic stem cells as a template. .. The targeting vector allowed the FLP recombinase-mediated excision of the neomycin selection cassette (gift of R. Fässler).

Article Title: Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis
Article Snippet: .. Promoter cloning The DNA fragment (3.1 kb), containing the MLK3 promoter was amplified from the HEK293 genomic DNA using the Expand High Fidelity PCR system (Roche Applied Science) and designated MLK3-P1. .. Five different deletion fragments (Figure ) were also generated using MLK3-P1 as a template and sub-cloned into pGL4.16 [luc2CP/Hygro] vector (Promega).

Article Title: The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A
Article Snippet: .. PCR was performed with the oligonucleotide designed to amplify the linear pMT2A-Luc altered the imperfect XRE2: Fwd, 5′-CACGaaaccGGCACCCAGCACC-3′ and Rev, 5′CGCCTCCGCCGTGTGCACAG-3′ using Expand High Fidelity PCR System (Roche Applied Science, Mannheim, Germany). .. This linear PCR product was phosphorylated and ligated itself after digestion of the template plasmid with Dpn I. AHR and GR expression plasmids were prepared as follows.

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: .. Generation of the 2KbHbp1P_pHSP68_LacZ mouse lines The 2 kb Hbp1 proximal promoter region was amplified from C57Bl/6 genomic DNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) using primers Hbp1P2kb-F and Hbp1P2kb-R ( ). .. The region included -2121 bp to +1 bp relative to the Hbp1 transcription start site and was cloned into a modified pBluescript vector containing LacZ driven by the minimal HSP68 promoter.

Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine
Article Snippet: .. Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI). .. The forward primer contained a XhoI restriction site, and the reverse primer contained a SacI restriction site for cloning into the XhoI/SacI sites in the multiple cloning site of the pBigT plasmid to create pBigT-Gata4 ( A ).

Article Title: Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment
Article Snippet: .. Constructs The mouse and human Use1 UTR was amplified using the Expand High Fidelity PCR System (Roche), a forward primer harboring an XhoI site and T7 promoter, and a reverse primer containing a NcoI site. .. PCR products were ligated in pCR2.1 (Invitrogen) and pGL2-basic.

Article Title: Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat
Article Snippet: .. Gene fragments for insertion into BSMV were PCR amplified using Expand High Fidelity PCR System (Roche Diagnostics, Mannheim, Germany) with the primers shown in Additional file . ..

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
Article Snippet: .. Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions. .. Genomic DNA used for PCRs was isolated from bacterial strains using the Wizard genomic DNA purification kit (Promega).

Article Title: Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues
Article Snippet: .. Generation and analysis of dronc reporter lacZ lines 1.64 kb and 1.33 kb of the dronc promoter were PCR amplified from Drosophila genomic DNA using Expand High Fidelity PCR System (Roche) with the primer sets DrPrF3cBglII (5′ CCG AGA TCT ATG TAC GTT ATG TTA TAG TAA GTG TA 3′); DrPrR1BglII (5′ CGG AGA TCT CCG GAT ATG GCT TCC ACG CGT 3′) and DrPrF3eBglII (5′ CGA AGA TCT AAT TGT GTA CAA CTA AAG GAA 3′); DrPrR1BglII, respectively. .. PCR products were cloned into pGem-T easy (Promega), and then subcloned into the BglII site of the p-element transformation lacZ reporter vector pCaSpeR-NLSlacZ (provided by Carl Thummel, Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT) after BglII digestion.

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: .. The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche). .. The following primer pairs were used to amplify the homology arms: 5′-gtc gac aga agt tct cgg agc ggg tgc tga act-3′, 5′-atc ctc cat gac agt tat ctg ca-3′ (for 5′ homology arm); 5′-tag gat cca caa agt cac aga gcc act ttc a-3′, 5′-tag gat ccc ctc tga ctt att ctt gct ct tag-3′ (for 3′ homology arm).

Article Title: HIV Restriction by APOBEC3 in Humanized Mice
Article Snippet: .. Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche). .. All PCR primers amplify both HIV-1JR-CSF and HIV-1LAI and were designed to anneal in regions with the fewest possible putative APOBEC3 deamination sites to avoid potential primer mismatch due to APOBEC3 induced mutagenesis.

Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors
Article Snippet: .. Briefly, probes were made by PCR from 3D7 WT gDNA for gfp, msp2, SSUD and SSU-Api using Expand High Fidelity PCR System (Roche, 3300226) with 0.3 M primers in a 50 μl reaction. .. The cycling parameters were as follows: 95 °C for 45 sec, 55 °C for 45 s, 64 °C for 1 min, 30 cycles.

DNA Sequencing:

Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis
Article Snippet: Paragraph title: WHV DNA sequencing. ... In selected cases, the full-length WHV DNA was amplified with primers PPC1 (1908-1926) and XPC (1891-1907) located at the WHV nick region, using the Expand High Fidelity PCR System (Roche Diagnostics, Laval, Quebec, Canada) and conditions described previously ( ).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
Article Snippet: .. Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions. .. Genomic DNA used for PCRs was isolated from bacterial strains using the Wizard genomic DNA purification kit (Promega).

Injection:

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells
Article Snippet: The flanks and “deletable” fragment of the Ebf1 gene were PCR-cloned with the Expand High-Fidelity PCR system (Roche) using genomic DNA from D3V embryonic stem cells as a template. .. Chimeras were obtained after injection of the targeted embryonic D3V stem cell clones into blastocysts derived from C57BL/6 mice.

Cellular Antioxidant Activity Assay:

Article Title: Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues
Article Snippet: .. Generation and analysis of dronc reporter lacZ lines 1.64 kb and 1.33 kb of the dronc promoter were PCR amplified from Drosophila genomic DNA using Expand High Fidelity PCR System (Roche) with the primer sets DrPrF3cBglII (5′ CCG AGA TCT ATG TAC GTT ATG TTA TAG TAA GTG TA 3′); DrPrR1BglII (5′ CGG AGA TCT CCG GAT ATG GCT TCC ACG CGT 3′) and DrPrF3eBglII (5′ CGA AGA TCT AAT TGT GTA CAA CTA AAG GAA 3′); DrPrR1BglII, respectively. .. PCR products were cloned into pGem-T easy (Promega), and then subcloned into the BglII site of the p-element transformation lacZ reporter vector pCaSpeR-NLSlacZ (provided by Carl Thummel, Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT) after BglII digestion.

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche). .. The following primer pairs were used to amplify the homology arms: 5′-gtc gac aga agt tct cgg agc ggg tgc tga act-3′, 5′-atc ctc cat gac agt tat ctg ca-3′ (for 5′ homology arm); 5′-tag gat cca caa agt cac aga gcc act ttc a-3′, 5′-tag gat ccc ctc tga ctt att ctt gct ct tag-3′ (for 3′ homology arm).

Fluorescence:

Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors
Article Snippet: We generated fluorescence-labeled probes (Fluorescein) for the gfp, msp2, SSUD and SSU-Api genes using the Label IT Nucleic Acid Labeling kit according to the instructions of the manufacturer (Mirus, MIR 3225). .. Briefly, probes were made by PCR from 3D7 WT gDNA for gfp, msp2, SSUD and SSU-Api using Expand High Fidelity PCR System (Roche, 3300226) with 0.3 M primers in a 50 μl reaction.

Mutagenesis:

Article Title: Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis
Article Snippet: Promoter cloning The DNA fragment (3.1 kb), containing the MLK3 promoter was amplified from the HEK293 genomic DNA using the Expand High Fidelity PCR system (Roche Applied Science) and designated MLK3-P1. .. Promoter with mutations (single or multiple) were generated using QuickChange Multi-Site Directed Mutagenesis kit (Agilent technologies).

Article Title: The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A
Article Snippet: The mutant MT2A imperfect XRE2 reporter plasmid was prepared using the following method. .. PCR was performed with the oligonucleotide designed to amplify the linear pMT2A-Luc altered the imperfect XRE2: Fwd, 5′-CACGaaaccGGCACCCAGCACC-3′ and Rev, 5′CGCCTCCGCCGTGTGCACAG-3′ using Expand High Fidelity PCR System (Roche Applied Science, Mannheim, Germany).

Article Title: Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment
Article Snippet: Constructs The mouse and human Use1 UTR was amplified using the Expand High Fidelity PCR System (Roche), a forward primer harboring an XhoI site and T7 promoter, and a reverse primer containing a NcoI site. .. The plasmids ΔSS, Δ GGGG and Δ AGGGCGGA were generated with the QuickChange Site-Direct Mutagenesis Kit (Stratagene).

Article Title: HIV Restriction by APOBEC3 in Humanized Mice
Article Snippet: Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche). .. All PCR primers amplify both HIV-1JR-CSF and HIV-1LAI and were designed to anneal in regions with the fewest possible putative APOBEC3 deamination sites to avoid potential primer mismatch due to APOBEC3 induced mutagenesis.

Isolation:

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions. .. Genomic DNA used for PCRs was isolated from bacterial strains using the Wizard genomic DNA purification kit (Promega).

Article Title: HIV Restriction by APOBEC3 in Humanized Mice
Article Snippet: Viral RNA was isolated from plasma using QIAamp viral RNA columns (Qiagen) according to the manufacture's protocol including an optional treatment with RNase-free DNase (Qiagen) during extraction and cDNA was generated using Superscript III Reverse Transcriptase (Invitrogen). .. Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche).

Size-exclusion Chromatography:

Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors
Article Snippet: Briefly, probes were made by PCR from 3D7 WT gDNA for gfp, msp2, SSUD and SSU-Api using Expand High Fidelity PCR System (Roche, 3300226) with 0.3 M primers in a 50 μl reaction. .. The cycling parameters were as follows: 95 °C for 45 sec, 55 °C for 45 s, 64 °C for 1 min, 30 cycles.

Labeling:

Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors
Article Snippet: We generated fluorescence-labeled probes (Fluorescein) for the gfp, msp2, SSUD and SSU-Api genes using the Label IT Nucleic Acid Labeling kit according to the instructions of the manufacturer (Mirus, MIR 3225). .. Briefly, probes were made by PCR from 3D7 WT gDNA for gfp, msp2, SSUD and SSU-Api using Expand High Fidelity PCR System (Roche, 3300226) with 0.3 M primers in a 50 μl reaction.

Mouse Assay:

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells
Article Snippet: Paragraph title: Generation of Ebf1+/fl mice ... The flanks and “deletable” fragment of the Ebf1 gene were PCR-cloned with the Expand High-Fidelity PCR system (Roche) using genomic DNA from D3V embryonic stem cells as a template.

Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine
Article Snippet: .. Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI). .. The forward primer contained a XhoI restriction site, and the reverse primer contained a SacI restriction site for cloning into the XhoI/SacI sites in the multiple cloning site of the pBigT plasmid to create pBigT-Gata4 ( A ).

Sequencing:

Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine
Article Snippet: .. Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI). .. The forward primer contained a XhoI restriction site, and the reverse primer contained a SacI restriction site for cloning into the XhoI/SacI sites in the multiple cloning site of the pBigT plasmid to create pBigT-Gata4 ( A ).

Article Title: Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment
Article Snippet: Constructs The mouse and human Use1 UTR was amplified using the Expand High Fidelity PCR System (Roche), a forward primer harboring an XhoI site and T7 promoter, and a reverse primer containing a NcoI site. .. In the ΔSS the AAGAUGG sequence around the start codon was changed into a NcoI site: ACCAUGG .

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche). .. PCR products were subcloned into the TOPO® vector (Invitrogen) and confirmed by DNA nucleotide sequencing.

Quantitative RT-PCR:

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
Article Snippet: .. Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions. .. Genomic DNA used for PCRs was isolated from bacterial strains using the Wizard genomic DNA purification kit (Promega).

Nested PCR:

Article Title: HIV Restriction by APOBEC3 in Humanized Mice
Article Snippet: .. Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche). .. All PCR primers amplify both HIV-1JR-CSF and HIV-1LAI and were designed to anneal in regions with the fewest possible putative APOBEC3 deamination sites to avoid potential primer mismatch due to APOBEC3 induced mutagenesis.

Activated Clotting Time Assay:

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche). .. The following primer pairs were used to amplify the homology arms: 5′-gtc gac aga agt tct cgg agc ggg tgc tga act-3′, 5′-atc ctc cat gac agt tat ctg ca-3′ (for 5′ homology arm); 5′-tag gat cca caa agt cac aga gcc act ttc a-3′, 5′-tag gat ccc ctc tga ctt att ctt gct ct tag-3′ (for 3′ homology arm).

In Situ Hybridization:

Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors
Article Snippet: Paragraph title: EV fluorescent in situ hybridization ... Briefly, probes were made by PCR from 3D7 WT gDNA for gfp, msp2, SSUD and SSU-Api using Expand High Fidelity PCR System (Roche, 3300226) with 0.3 M primers in a 50 μl reaction.

Plasmid Preparation:

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: HBP1-MYC constructs Truncated Hbp1 cDNA constructs were amplified from 12.5 dpc testis cDNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) with the common forward primer All-Hbp1-MYC-F containing a BamHI restriction site and individual reverse primers containing an XhoI restriction site, FlHbp1-MYC-R and ΔHbp1-MYC-R ( ). .. These fragments were cloned into a modified pcDNA3.0 vector generating an N-terminal MYC-tagged protein and sequenced for verification.

Article Title: Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells
Article Snippet: Expression Vectors Plasmid DNA encoding wild type (WT) β-catenin was used as previously described and DNA encoding wild type Snail was a kind gift from Dr. Mien-Chie Hung , . .. Cadherin-11 3′-UTR fragment was cloned from genomic DNA obtained from MDA-MB-231 cells using Expand High Fidelity PCR System (11732641001, Roche, Indianapolis, IN) according to the manufacturer's protocol.

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells
Article Snippet: The flanks and “deletable” fragment of the Ebf1 gene were PCR-cloned with the Expand High-Fidelity PCR system (Roche) using genomic DNA from D3V embryonic stem cells as a template. .. The targeting vector allowed the FLP recombinase-mediated excision of the neomycin selection cassette (gift of R. Fässler).

Article Title: Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis
Article Snippet: Promoter cloning The DNA fragment (3.1 kb), containing the MLK3 promoter was amplified from the HEK293 genomic DNA using the Expand High Fidelity PCR system (Roche Applied Science) and designated MLK3-P1. .. Five different deletion fragments (Figure ) were also generated using MLK3-P1 as a template and sub-cloned into pGL4.16 [luc2CP/Hygro] vector (Promega).

Article Title: The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A
Article Snippet: Paragraph title: Plasmid construction. ... PCR was performed with the oligonucleotide designed to amplify the linear pMT2A-Luc altered the imperfect XRE2: Fwd, 5′-CACGaaaccGGCACCCAGCACC-3′ and Rev, 5′CGCCTCCGCCGTGTGCACAG-3′ using Expand High Fidelity PCR System (Roche Applied Science, Mannheim, Germany).

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: Generation of the 2KbHbp1P_pHSP68_LacZ mouse lines The 2 kb Hbp1 proximal promoter region was amplified from C57Bl/6 genomic DNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) using primers Hbp1P2kb-F and Hbp1P2kb-R ( ). .. The region included -2121 bp to +1 bp relative to the Hbp1 transcription start site and was cloned into a modified pBluescript vector containing LacZ driven by the minimal HSP68 promoter.

Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine
Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI). .. The forward primer contained a XhoI restriction site, and the reverse primer contained a SacI restriction site for cloning into the XhoI/SacI sites in the multiple cloning site of the pBigT plasmid to create pBigT-Gata4 ( A ).

Article Title: Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues
Article Snippet: Generation and analysis of dronc reporter lacZ lines 1.64 kb and 1.33 kb of the dronc promoter were PCR amplified from Drosophila genomic DNA using Expand High Fidelity PCR System (Roche) with the primer sets DrPrF3cBglII (5′ CCG AGA TCT ATG TAC GTT ATG TTA TAG TAA GTG TA 3′); DrPrR1BglII (5′ CGG AGA TCT CCG GAT ATG GCT TCC ACG CGT 3′) and DrPrF3eBglII (5′ CGA AGA TCT AAT TGT GTA CAA CTA AAG GAA 3′); DrPrR1BglII, respectively. .. PCR products were cloned into pGem-T easy (Promega), and then subcloned into the BglII site of the p-element transformation lacZ reporter vector pCaSpeR-NLSlacZ (provided by Carl Thummel, Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT) after BglII digestion.

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: Paragraph title: Construction of rat p53 gene targeting vector ... The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche).

Dominant Negative Mutation:

Article Title: Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and ?-Catenin in Prostate and Breast Cancer Cells
Article Snippet: Dominant negative (DN) Snail was generated by PCR from the wild type Snail construct using primers described in Yamasaki et al. forward: ( 5′-CGGGATCCACTATGGCCTTCAACTGCAAATACTG-3′ ) and reverse: ( 5′-CGCTCGAGGCGGGGACATCCTGAGCA-3′ ) and cloned into pCMV-Tag2 (Stratagene, La Jolla, CA) using BamHI and XhoI restriction sites . .. Cadherin-11 3′-UTR fragment was cloned from genomic DNA obtained from MDA-MB-231 cells using Expand High Fidelity PCR System (11732641001, Roche, Indianapolis, IN) according to the manufacturer's protocol.

Selection:

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells
Article Snippet: The flanks and “deletable” fragment of the Ebf1 gene were PCR-cloned with the Expand High-Fidelity PCR system (Roche) using genomic DNA from D3V embryonic stem cells as a template. .. The targeting vector allowed the FLP recombinase-mediated excision of the neomycin selection cassette (gift of R. Fässler).

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: We then constructed a p53 gene targeting vector using the CAG promoter to drive the expression of the EGFP-IRES-Pac selection cassette. .. The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche).

In Situ:

Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors
Article Snippet: EV fluorescent in situ hybridization The EV-FISH preparation was developed as a new protocol for gene in situ detection in EVs. .. Briefly, probes were made by PCR from 3D7 WT gDNA for gfp, msp2, SSUD and SSU-Api using Expand High Fidelity PCR System (Roche, 3300226) with 0.3 M primers in a 50 μl reaction.

Transgenic Assay:

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: Generation of the 2KbHbp1P_pHSP68_LacZ mouse lines The 2 kb Hbp1 proximal promoter region was amplified from C57Bl/6 genomic DNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) using primers Hbp1P2kb-F and Hbp1P2kb-R ( ). .. Transgenic mouse lines were produced by standard methods [ ].

Produced:

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile
Article Snippet: Generation of the 2KbHbp1P_pHSP68_LacZ mouse lines The 2 kb Hbp1 proximal promoter region was amplified from C57Bl/6 genomic DNA using the Expand High Fidelity PCR System (Roche, Indianapolis, USA) using primers Hbp1P2kb-F and Hbp1P2kb-R ( ). .. Transgenic mouse lines were produced by standard methods [ ].

Mobility Shift:

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
Article Snippet: .. Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions. .. Genomic DNA used for PCRs was isolated from bacterial strains using the Wizard genomic DNA purification kit (Promega).

DNA Purification:

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions. .. Genomic DNA used for PCRs was isolated from bacterial strains using the Wizard genomic DNA purification kit (Promega).

CTG Assay:

Article Title: Production of p53 gene knockout rats by homologous recombination in embryonic stem cells
Article Snippet: The 5′ and 3′ homology arms were amplified from DA rat genomic DNA using Expand High Fidelity PCR System (Roche). .. The following primer pairs were used to amplify the homology arms: 5′-gtc gac aga agt tct cgg agc ggg tgc tga act-3′, 5′-atc ctc cat gac agt tat ctg ca-3′ (for 5′ homology arm); 5′-tag gat cca caa agt cac aga gcc act ttc a-3′, 5′-tag gat ccc ctc tga ctt att ctt gct ct tag-3′ (for 3′ homology arm).

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    Roche expand high fidelity pcr system
    Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in <t>GATA4-expressing</t> (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. <t>RT-PCR</t> was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI ( ROSA26 lnlG4/+ Villin-Cre ) and control ( ROSA26 lnlG4/+ ) mice and in jejunal epithelial cells from Gata4 cKO ( Gata4 loxP/loxP Villin-Cre ) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold . Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. * P ≤ .05. ** P ≤ .01.
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    Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in GATA4-expressing (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. RT-PCR was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI ( ROSA26 lnlG4/+ Villin-Cre ) and control ( ROSA26 lnlG4/+ ) mice and in jejunal epithelial cells from Gata4 cKO ( Gata4 loxP/loxP Villin-Cre ) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold . Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. * P ≤ .05. ** P ≤ .01.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in GATA4-expressing (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. RT-PCR was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI ( ROSA26 lnlG4/+ Villin-Cre ) and control ( ROSA26 lnlG4/+ ) mice and in jejunal epithelial cells from Gata4 cKO ( Gata4 loxP/loxP Villin-Cre ) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold . Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. * P ≤ .05. ** P ≤ .01.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Mouse Assay

    Duodenal and jejunal epithelial cells in Gata4 cKI mice express normal levels of GATA4. ( A ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in duodenal and jejunal epithelium of Gata4 cKI mice at similar staining intensity compared with controls. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( B ) qRT-PCR showed that Gata4 mRNA was unchanged in epithelial cells of the duodenum and jejunum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with control mice ( ROSA26 lnlG4/+ ) (n = 3 per genotype; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test. ( C ) Immunoblot analysis of nuclear extracts from duodenal and jejunal epithelial cells of control and Gata4 cKI mice was used to quantify GATA4 protein (n = 3 per genotype). To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression was unchanged in duodenum and jejunum of Gata4 cKI animals compared with control. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Duodenal and jejunal epithelial cells in Gata4 cKI mice express normal levels of GATA4. ( A ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in duodenal and jejunal epithelium of Gata4 cKI mice at similar staining intensity compared with controls. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( B ) qRT-PCR showed that Gata4 mRNA was unchanged in epithelial cells of the duodenum and jejunum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with control mice ( ROSA26 lnlG4/+ ) (n = 3 per genotype; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test. ( C ) Immunoblot analysis of nuclear extracts from duodenal and jejunal epithelial cells of control and Gata4 cKI mice was used to quantify GATA4 protein (n = 3 per genotype). To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression was unchanged in duodenum and jejunum of Gata4 cKI animals compared with control. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Mouse Assay, Immunohistochemistry, Staining, Quantitative RT-PCR, Expressing, Software, Binding Assay, Molecular Weight, Marker

    Enterohepatic signaling is altered in animals expressing GATA4 in the ileum. ( A ) Immunohistochemistry for SLC10A2 (brown stain) shows SLC10A2 protein lining the brush border of control ileum (n = 7, upper panel). In contrast, SLC10A2 staining was faint to nearly absent along the brush border of ileum from Gata4 cKI mice (n = 14, 7 of 14 faint SLC10A2 staining, middle panel , and 7 of 14 low to no SLC10A2 staining, lower panel ). Immunohistochemistry from 2 independent Gata4 cKI mice is shown as representative of the 2 types of SLC10A2 staining observed. ( B ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of SLC10A2 protein. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. SLC10A2 protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. SLC10A2 expression in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 9% of the level observed in control ileum ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Arrowhead indicates the SLC10A2 band measured. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( C ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of OSTα and OSTβ proteins. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. OSTα and OSTβ protein levels were normalized to GAPDH levels. Expression of OSTα and OSTβ proteins in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 26% and 19% of the level observed in control ileum ( ROSA26 lnlG4/+ ), respectively (n = 3 animals per genotype). Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) qRT-PCR shows increased Cyp7a1 expression in liver from Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) compared with control mice ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars represent SEM. * P ≤ .05.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Enterohepatic signaling is altered in animals expressing GATA4 in the ileum. ( A ) Immunohistochemistry for SLC10A2 (brown stain) shows SLC10A2 protein lining the brush border of control ileum (n = 7, upper panel). In contrast, SLC10A2 staining was faint to nearly absent along the brush border of ileum from Gata4 cKI mice (n = 14, 7 of 14 faint SLC10A2 staining, middle panel , and 7 of 14 low to no SLC10A2 staining, lower panel ). Immunohistochemistry from 2 independent Gata4 cKI mice is shown as representative of the 2 types of SLC10A2 staining observed. ( B ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of SLC10A2 protein. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. SLC10A2 protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. SLC10A2 expression in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 9% of the level observed in control ileum ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Arrowhead indicates the SLC10A2 band measured. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( C ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of OSTα and OSTβ proteins. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. OSTα and OSTβ protein levels were normalized to GAPDH levels. Expression of OSTα and OSTβ proteins in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 26% and 19% of the level observed in control ileum ( ROSA26 lnlG4/+ ), respectively (n = 3 animals per genotype). Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) qRT-PCR shows increased Cyp7a1 expression in liver from Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) compared with control mice ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars represent SEM. * P ≤ .05.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Expressing, Immunohistochemistry, Staining, Mouse Assay, Software, Molecular Weight, Marker, Quantitative RT-PCR

    Expression of jejunal and duodenal transcripts is unchanged in Gata4 cKI animals. ( A ) qRT-PCR was used to determine transcript abundance of the 10 jejunal-enriched transcripts, identified as having enriched GATA4 binding by bio-ChIP–PCR ( Figure 7 ) in jejunal epithelial cells from control and Gata4 cKI mice. ( B ) qRT-PCR was used to determine transcript abundance of 4 duodenal transcripts in duodenal epithelial cells from control and Gata4 cKI mice. ( A and B ) Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments (n = 3 per genotype). Error bars represent SEM. P values were determined by 2-sample Student t test.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Expression of jejunal and duodenal transcripts is unchanged in Gata4 cKI animals. ( A ) qRT-PCR was used to determine transcript abundance of the 10 jejunal-enriched transcripts, identified as having enriched GATA4 binding by bio-ChIP–PCR ( Figure 7 ) in jejunal epithelial cells from control and Gata4 cKI mice. ( B ) qRT-PCR was used to determine transcript abundance of 4 duodenal transcripts in duodenal epithelial cells from control and Gata4 cKI mice. ( A and B ) Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments (n = 3 per genotype). Error bars represent SEM. P values were determined by 2-sample Student t test.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Mouse Assay

    Representative autoradiographs of bio-ChIP–PCR. Bio-ChIP–PCR was used to evaluate GATA4 occupancy at predicted binding sites in the 26 high-confidence direct targets we identified and in 7 negative controls ( Alb, Cdk4, Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ). GATA4 occupied chromatin was isolated by performing streptavidin pull-down with chromatin from jejunal epithelial cells of GATA4-FlagBio/BirA or GATA4-WT/BirA mice. As representative data, PCR with chromatin from 2 mice per genotype is shown here. Input PCR confirmed that equivalent chromatin amounts were used in pull-downs. In all, 6 mice per genotype were assayed by bio-ChIP–PCR.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Representative autoradiographs of bio-ChIP–PCR. Bio-ChIP–PCR was used to evaluate GATA4 occupancy at predicted binding sites in the 26 high-confidence direct targets we identified and in 7 negative controls ( Alb, Cdk4, Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ). GATA4 occupied chromatin was isolated by performing streptavidin pull-down with chromatin from jejunal epithelial cells of GATA4-FlagBio/BirA or GATA4-WT/BirA mice. As representative data, PCR with chromatin from 2 mice per genotype is shown here. Input PCR confirmed that equivalent chromatin amounts were used in pull-downs. In all, 6 mice per genotype were assayed by bio-ChIP–PCR.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Isolation, Mouse Assay

    Gata4 conditional knock-in mice express GATA4 in the ileum. ( A ) Schematic illustrating the strategy used to generate a conditional Gata4 knock-in mouse line. The coding sequence of the mouse Gata4 gene was amplified by PCR and inserted into XhoI/SacI sites in the multiple cloning site (MCS) of pBig-T to generate pBigT- Gata4 . The targeting cassette consisting of an adenoviral splice acceptor (SA), a loxP flanked phosphoglycerate kinase (PGK) promoter-neomycin resistance gene (Neo) and 3×SV40 polyadenylation sequence (pA) sequence ( loxP -PGK-Neo-3×SV40pA- loxP , LNL), the Gata4 coding sequence, and a bovine growth hormone polyadenylation (pA) sequence was excised from pBigT- Gata4 with PacI/AscI and inserted into the PacI/AscI sites in pROSA26PA to create pROSA26PA- Gata4 . Homologous recombination between pROSA26PA- Gata4 and the endogenous ROSA26 locus in mouse R1 embryonic stem cells yielded the targeted locus Gt(ROSA)26Sor tm1(Gata4)Bat , designated ROSA26 lnlG4 . After Cre recombination to excise the LNL cassette, Gata4 is expressed. BamHI ( B ) and EcoRV ( E ) restriction sites used for Southern blot analysis, the position of Southern blot probes, and relevant BamHI and EcoRV restriction digest fragments identified by Southern blot are shown. Arrows mark sites of genotyping primers ( Table 1 , primers). ( B ) Southern blot analysis confirmed germline transmission of the ROSA26 lnlG4 allele. Representative Southern blot analysis of EcoRV or BamHI digested genomic DNA harvested from a wild-type mouse (ROSA26 +/+ ) or a mouse heterozygous for the modified ROSA26 allele ( ROSA26 lnlG4/+ ). We observed the expected fragments representing the wild-type and modified alleles ( EcoRV digest, 11.5-kb wild-type allele and 4.0-kb modified allele; BamHI digest, 5.8-kb wild-type allele and 4.7-kb modified allele). ( C ) qRT-PCR showed that Gata4 mRNA was induced in ileum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with ileum of control mice ( ROSA26 lnlG4/+ ). Gata6 mRNA remained unchanged in the ileum of Gata4 cKI mice compared with controls (n = ileum of 5 control and 6 Gata4 cKI animals; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in ileal epithelium of Gata4 cKI mice and in the jejunal epithelium of control mice whereas GATA4 protein was absent from ileal epithelium of control mice. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( E ) Immunoblot analysis of nuclear extracts from jejunal and ileal epithelial cells of control mice and from ileal epithelial cells of Gata4 cKI mice was used to quantify GATA4 protein in ileum of Gata4 cKI mice and to compare GATA4 abundance between control jejunum and GATA4-expressing ileum. The blot shown contains nuclear protein extracts from 3 control and 3 Gata4 cKI animals and is representative of analysis of more than 24 control and Gata4 cKI animals. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression in ileum of Gata4 cKI mice was 27% the level observed in control jejunum. Molecular weight marker locations are indicated. Error bars show SEM. P values determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .001.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Gata4 conditional knock-in mice express GATA4 in the ileum. ( A ) Schematic illustrating the strategy used to generate a conditional Gata4 knock-in mouse line. The coding sequence of the mouse Gata4 gene was amplified by PCR and inserted into XhoI/SacI sites in the multiple cloning site (MCS) of pBig-T to generate pBigT- Gata4 . The targeting cassette consisting of an adenoviral splice acceptor (SA), a loxP flanked phosphoglycerate kinase (PGK) promoter-neomycin resistance gene (Neo) and 3×SV40 polyadenylation sequence (pA) sequence ( loxP -PGK-Neo-3×SV40pA- loxP , LNL), the Gata4 coding sequence, and a bovine growth hormone polyadenylation (pA) sequence was excised from pBigT- Gata4 with PacI/AscI and inserted into the PacI/AscI sites in pROSA26PA to create pROSA26PA- Gata4 . Homologous recombination between pROSA26PA- Gata4 and the endogenous ROSA26 locus in mouse R1 embryonic stem cells yielded the targeted locus Gt(ROSA)26Sor tm1(Gata4)Bat , designated ROSA26 lnlG4 . After Cre recombination to excise the LNL cassette, Gata4 is expressed. BamHI ( B ) and EcoRV ( E ) restriction sites used for Southern blot analysis, the position of Southern blot probes, and relevant BamHI and EcoRV restriction digest fragments identified by Southern blot are shown. Arrows mark sites of genotyping primers ( Table 1 , primers). ( B ) Southern blot analysis confirmed germline transmission of the ROSA26 lnlG4 allele. Representative Southern blot analysis of EcoRV or BamHI digested genomic DNA harvested from a wild-type mouse (ROSA26 +/+ ) or a mouse heterozygous for the modified ROSA26 allele ( ROSA26 lnlG4/+ ). We observed the expected fragments representing the wild-type and modified alleles ( EcoRV digest, 11.5-kb wild-type allele and 4.0-kb modified allele; BamHI digest, 5.8-kb wild-type allele and 4.7-kb modified allele). ( C ) qRT-PCR showed that Gata4 mRNA was induced in ileum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with ileum of control mice ( ROSA26 lnlG4/+ ). Gata6 mRNA remained unchanged in the ileum of Gata4 cKI mice compared with controls (n = ileum of 5 control and 6 Gata4 cKI animals; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in ileal epithelium of Gata4 cKI mice and in the jejunal epithelium of control mice whereas GATA4 protein was absent from ileal epithelium of control mice. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( E ) Immunoblot analysis of nuclear extracts from jejunal and ileal epithelial cells of control mice and from ileal epithelial cells of Gata4 cKI mice was used to quantify GATA4 protein in ileum of Gata4 cKI mice and to compare GATA4 abundance between control jejunum and GATA4-expressing ileum. The blot shown contains nuclear protein extracts from 3 control and 3 Gata4 cKI animals and is representative of analysis of more than 24 control and Gata4 cKI animals. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression in ileum of Gata4 cKI mice was 27% the level observed in control jejunum. Molecular weight marker locations are indicated. Error bars show SEM. P values determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .001.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Knock-In, Mouse Assay, Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, Southern Blot, Transmission Assay, Modification, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing, Software, Binding Assay, Molecular Weight, Marker

    GATA4 occupies sites in jejunal- and ileal-enriched genes, suggesting GATA4 directly regulates expression of jejunal- and ileal-enriched genes in the jejunum to define jejunal enterocyte identity. Bio-ChIP–PCR showed GATA4 enrichment at GATA4 binding sites within genes expressed in jejunum ( top panel ) and within genes repressed in jejunum ( middle panel ). No GATA4 enrichment was observed at sites lacking GATA4 bio-ChIP-seq binding sites ( Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ) or in genes identified as GATA4 targets in other tissues but that are either equivalently expressed in ileum of control and Gata4 cKI mice ( Cdk4 ) or absent in ileum of control and Gata4 cKI mice ( Alb ) ( bottom panel ). Audioradiographic band intensity was measured using a Storm820 Phosphor Imager and ImageQuant software. Representative autoradiographs for each site assayed are shown in Figure 7 . Enrichment per sample was normalized to input (n = 6 Gata4 flbio/flbio ::ROSA26 BirA/BirA mice, designated GATA4-FlagBio/BirA , and 6 ROSA26 BirA/BirA mice, designated GATA4-WT/BirA ). Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .005. P values > .05 are listed on graphs. GATA4 occupancy at the binding sites in the Slc10a2 gene ( Slc10a2 _1 and Slc10a2 _2) was analyzed previously by qPCR. 38

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: GATA4 occupies sites in jejunal- and ileal-enriched genes, suggesting GATA4 directly regulates expression of jejunal- and ileal-enriched genes in the jejunum to define jejunal enterocyte identity. Bio-ChIP–PCR showed GATA4 enrichment at GATA4 binding sites within genes expressed in jejunum ( top panel ) and within genes repressed in jejunum ( middle panel ). No GATA4 enrichment was observed at sites lacking GATA4 bio-ChIP-seq binding sites ( Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ) or in genes identified as GATA4 targets in other tissues but that are either equivalently expressed in ileum of control and Gata4 cKI mice ( Cdk4 ) or absent in ileum of control and Gata4 cKI mice ( Alb ) ( bottom panel ). Audioradiographic band intensity was measured using a Storm820 Phosphor Imager and ImageQuant software. Representative autoradiographs for each site assayed are shown in Figure 7 . Enrichment per sample was normalized to input (n = 6 Gata4 flbio/flbio ::ROSA26 BirA/BirA mice, designated GATA4-FlagBio/BirA , and 6 ROSA26 BirA/BirA mice, designated GATA4-WT/BirA ). Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .005. P values > .05 are listed on graphs. GATA4 occupancy at the binding sites in the Slc10a2 gene ( Slc10a2 _1 and Slc10a2 _2) was analyzed previously by qPCR. 38

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Expressing, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Mouse Assay, Software, Real-time Polymerase Chain Reaction

    Identification of WHV RNA sequences in naive and mitogen-stimulated PBMCs in offspring born to mothers convalescent from viral hepatitis. Total RNA was extracted from ( a ) unstimulated PBMCs from 4B/M offspring obtained at 18, 30, and 32 months after birth and from ( b ) unstimulated (–ConA) and Con A–stimulated (+ConA) PBMCs from 11D/F offspring at 34.5 months after birth. RNA isolated from Con A–stimulated PBMCs of a WHV-negative, healthy woodchuck (Control PBMCs + ConA) and from the spleen of a WHsAg-positive chronic WHV carrier (Positive control) was used as control. For all RT reactions, cDNA synthesis was carried out by RT (RT+) and the cDNA analyzed by nested PCR/Southern blot hybridization. Each RNA sample was subjected to the same reaction conditions in the absence of RT (RT–).

    Journal: Journal of Clinical Investigation

    Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

    doi:

    Figure Lengend Snippet: Identification of WHV RNA sequences in naive and mitogen-stimulated PBMCs in offspring born to mothers convalescent from viral hepatitis. Total RNA was extracted from ( a ) unstimulated PBMCs from 4B/M offspring obtained at 18, 30, and 32 months after birth and from ( b ) unstimulated (–ConA) and Con A–stimulated (+ConA) PBMCs from 11D/F offspring at 34.5 months after birth. RNA isolated from Con A–stimulated PBMCs of a WHV-negative, healthy woodchuck (Control PBMCs + ConA) and from the spleen of a WHsAg-positive chronic WHV carrier (Positive control) was used as control. For all RT reactions, cDNA synthesis was carried out by RT (RT+) and the cDNA analyzed by nested PCR/Southern blot hybridization. Each RNA sample was subjected to the same reaction conditions in the absence of RT (RT–).

    Article Snippet: In selected cases, the full-length WHV DNA was amplified with primers PPC1 (1908-1926) and XPC (1891-1907) located at the WHV nick region, using the Expand High Fidelity PCR System (Roche Diagnostics, Laval, Quebec, Canada) and conditions described previously ( ).

    Techniques: Isolation, Positive Control, Nested PCR, Southern Blot, Hybridization

    Detection of WHV cccDNA in selected WHV DNA–reactive liver, PBMCs, and lymphoid tissue samples from 3B/M, 4B/M, and 5C/F offspring. DNA was extracted from autopsy spleen and bone marrow of 3B/M; from liver biopsies collected at 19 and 31 months and PBMCs obtained at 34 and 41 months after birth from 4B/M; and from liver, spleen, and bone marrow collected at autopsy of 5C/F performed at 22 months of age. The PBMCs harvested from 4B/M at 41 months after birth were extensively washed and the cell surface treated with DNase and trypsin before DNA isolation. DNA samples (2 or 5 μg) were digested with mung bean endonuclease and subjected to nested PCR with primers amplifying the WHV gap-spanning region. DNA samples from a WHsAg-positive chronic carrier were included as positive controls; water instead of DNA and a mock sample extracted in parallel with test samples were used as negative controls. Positive samples showed the expected size of the amplified nucleotide fragments indicated on the left.

    Journal: Journal of Clinical Investigation

    Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

    doi:

    Figure Lengend Snippet: Detection of WHV cccDNA in selected WHV DNA–reactive liver, PBMCs, and lymphoid tissue samples from 3B/M, 4B/M, and 5C/F offspring. DNA was extracted from autopsy spleen and bone marrow of 3B/M; from liver biopsies collected at 19 and 31 months and PBMCs obtained at 34 and 41 months after birth from 4B/M; and from liver, spleen, and bone marrow collected at autopsy of 5C/F performed at 22 months of age. The PBMCs harvested from 4B/M at 41 months after birth were extensively washed and the cell surface treated with DNase and trypsin before DNA isolation. DNA samples (2 or 5 μg) were digested with mung bean endonuclease and subjected to nested PCR with primers amplifying the WHV gap-spanning region. DNA samples from a WHsAg-positive chronic carrier were included as positive controls; water instead of DNA and a mock sample extracted in parallel with test samples were used as negative controls. Positive samples showed the expected size of the amplified nucleotide fragments indicated on the left.

    Article Snippet: In selected cases, the full-length WHV DNA was amplified with primers PPC1 (1908-1926) and XPC (1891-1907) located at the WHV nick region, using the Expand High Fidelity PCR System (Roche Diagnostics, Laval, Quebec, Canada) and conditions described previously ( ).

    Techniques: DNA Extraction, Nested PCR, Amplification

    Effect of DNase digestion on WHV DNA–reactive particles circulating in an offspring born to a mother convalescent from viral hepatitis. Serum obtained at 32 months after birth from 4B/M offspring with WHV DNA expression in both liver and PBMCs; purified WHV virions; and Mnl I-digested recombinant WHV DNA were centrifuged through 15% sucrose over a 60% sucrose cushion, as described in Methods. Fifteen fractions collected from the bottom of each gradient were tested for WHV DNA by PCR, and those showing highest WHV DNA reactivity were pooled and were digested with DNase (D). Tested samples included pooled fractions 1–3 (bottom) for WHV virions, 1–5 (bottom) and 8–12 (top) for 4B/M serum, and 9–11 (top) for recombinant WHV DNA. As controls, samples of the same pooled fractions, but this time not treated with DNase (ND), were used. DNA extracted from each sample was tested for WHV S gene sequences by nested PCR and Southern blot hybridization.

    Journal: Journal of Clinical Investigation

    Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

    doi:

    Figure Lengend Snippet: Effect of DNase digestion on WHV DNA–reactive particles circulating in an offspring born to a mother convalescent from viral hepatitis. Serum obtained at 32 months after birth from 4B/M offspring with WHV DNA expression in both liver and PBMCs; purified WHV virions; and Mnl I-digested recombinant WHV DNA were centrifuged through 15% sucrose over a 60% sucrose cushion, as described in Methods. Fifteen fractions collected from the bottom of each gradient were tested for WHV DNA by PCR, and those showing highest WHV DNA reactivity were pooled and were digested with DNase (D). Tested samples included pooled fractions 1–3 (bottom) for WHV virions, 1–5 (bottom) and 8–12 (top) for 4B/M serum, and 9–11 (top) for recombinant WHV DNA. As controls, samples of the same pooled fractions, but this time not treated with DNase (ND), were used. DNA extracted from each sample was tested for WHV S gene sequences by nested PCR and Southern blot hybridization.

    Article Snippet: In selected cases, the full-length WHV DNA was amplified with primers PPC1 (1908-1926) and XPC (1891-1907) located at the WHV nick region, using the Expand High Fidelity PCR System (Roche Diagnostics, Laval, Quebec, Canada) and conditions described previously ( ).

    Techniques: Expressing, Purification, Recombinant, Polymerase Chain Reaction, Nested PCR, Southern Blot, Hybridization

    Analysis of WHV DNA expression in liver, PBMCs, and lymphoid tissues collected from 3B/M offspring. WHV gene sequences were identified by nested PCR using C and X gene–specific primers, followed by Southern blot hybridization of the amplified products to recombinant WHV DNA. Five micrograms of DNA extracted from liver samples collected at 6 months of age and at autopsy performed at 15 months after birth, and 1 μg DNA from PBMCs collected at 14.5 months of age and from spleen, lymph node, bone marrow, and skeletal muscle obtained at autopsy, were used for direct PCR amplification. Positive samples showed the expected molecular size of the amplified virus C and X gene fragments indicated on the left.

    Journal: Journal of Clinical Investigation

    Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

    doi:

    Figure Lengend Snippet: Analysis of WHV DNA expression in liver, PBMCs, and lymphoid tissues collected from 3B/M offspring. WHV gene sequences were identified by nested PCR using C and X gene–specific primers, followed by Southern blot hybridization of the amplified products to recombinant WHV DNA. Five micrograms of DNA extracted from liver samples collected at 6 months of age and at autopsy performed at 15 months after birth, and 1 μg DNA from PBMCs collected at 14.5 months of age and from spleen, lymph node, bone marrow, and skeletal muscle obtained at autopsy, were used for direct PCR amplification. Positive samples showed the expected molecular size of the amplified virus C and X gene fragments indicated on the left.

    Article Snippet: In selected cases, the full-length WHV DNA was amplified with primers PPC1 (1908-1926) and XPC (1891-1907) located at the WHV nick region, using the Expand High Fidelity PCR System (Roche Diagnostics, Laval, Quebec, Canada) and conditions described previously ( ).

    Techniques: Expressing, Nested PCR, Southern Blot, Hybridization, Amplification, Recombinant, Polymerase Chain Reaction

    WHV DNA expression in serum, liver, and lymphoid cells of 260/M woodchuck after inoculation with serum from liver WHV DNA–negative 3B/M offspring. Five micrograms of total DNA from liver biopsies collected at ∼2 months before (1) and 2 months after (2) inoculation and at autopsy (3), and 1 μg of DNA from autopsy PBMCs, isolated splenocytes, and bone marrow obtained at 3.5 months after inoculation or from 50 μL of autopsy serum, were tested for WHV DNA by nested PCR using WHV C gene–specific primers and hybridization to WHV DNA probe. DNA from serum of a WHsAg-positive chronic carrier was included as a positive control, and water instead of DNA and a mock sample extracted in parallel with test samples were used negative controls. Positive samples showed the expected 428-bp nucleotide fragment noted by arrowhead.

    Journal: Journal of Clinical Investigation

    Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

    doi:

    Figure Lengend Snippet: WHV DNA expression in serum, liver, and lymphoid cells of 260/M woodchuck after inoculation with serum from liver WHV DNA–negative 3B/M offspring. Five micrograms of total DNA from liver biopsies collected at ∼2 months before (1) and 2 months after (2) inoculation and at autopsy (3), and 1 μg of DNA from autopsy PBMCs, isolated splenocytes, and bone marrow obtained at 3.5 months after inoculation or from 50 μL of autopsy serum, were tested for WHV DNA by nested PCR using WHV C gene–specific primers and hybridization to WHV DNA probe. DNA from serum of a WHsAg-positive chronic carrier was included as a positive control, and water instead of DNA and a mock sample extracted in parallel with test samples were used negative controls. Positive samples showed the expected 428-bp nucleotide fragment noted by arrowhead.

    Article Snippet: In selected cases, the full-length WHV DNA was amplified with primers PPC1 (1908-1926) and XPC (1891-1907) located at the WHV nick region, using the Expand High Fidelity PCR System (Roche Diagnostics, Laval, Quebec, Canada) and conditions described previously ( ).

    Techniques: Expressing, Isolation, Nested PCR, Hybridization, Positive Control

    Detection of WHV DNA in serial serum samples from 4B/M offspring. Sera collected between 2 and 36 months after birth were tested for WHV DNA by nested PCR using WHV C, S, and X gene–specific primers, and the amplified products were analyzed by Southern blot hybridization to recombinant WHV DNA probe. DNA isolated from serum of a WHsAg-positive chronic carrier and water were used as positive control and negative control, respectively. Positive samples show the expected sizes (bp) of the amplified nucleotide fragments noted by arrowheads.

    Journal: Journal of Clinical Investigation

    Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

    doi:

    Figure Lengend Snippet: Detection of WHV DNA in serial serum samples from 4B/M offspring. Sera collected between 2 and 36 months after birth were tested for WHV DNA by nested PCR using WHV C, S, and X gene–specific primers, and the amplified products were analyzed by Southern blot hybridization to recombinant WHV DNA probe. DNA isolated from serum of a WHsAg-positive chronic carrier and water were used as positive control and negative control, respectively. Positive samples show the expected sizes (bp) of the amplified nucleotide fragments noted by arrowheads.

    Article Snippet: In selected cases, the full-length WHV DNA was amplified with primers PPC1 (1908-1926) and XPC (1891-1907) located at the WHV nick region, using the Expand High Fidelity PCR System (Roche Diagnostics, Laval, Quebec, Canada) and conditions described previously ( ).

    Techniques: Nested PCR, Amplification, Southern Blot, Hybridization, Recombinant, Isolation, Positive Control, Negative Control

    Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation, Plasmid Preparation

    Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    vif -deleted HIV-1 JR-CSF does not overcome APOBEC3 restriction in vivo . (A) Longitudinal analysis of plasma viral load in humanized mice intravenously infected with 9×10 4 TCIU of wild-type HIV-1 JR-CSF or 3.6×10 5 TCIU of HIV JR-CSF Δ vif . (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 17 viral DNA sequences amplified from the tissues of infected mice. Viral DNA from mice infected with HIV JR-CSF Δ vif had 25%–65% of G G sites mutated. (D) G to A mutational profile of all viral DNA from mice infected with HIV JR-CSF Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites. D4-D8, WT2-WT3, NSG-hu mice. D9-D11, NSG-BLT mice.

    Journal: PLoS Pathogens

    Article Title: HIV Restriction by APOBEC3 in Humanized Mice

    doi: 10.1371/journal.ppat.1003242

    Figure Lengend Snippet: vif -deleted HIV-1 JR-CSF does not overcome APOBEC3 restriction in vivo . (A) Longitudinal analysis of plasma viral load in humanized mice intravenously infected with 9×10 4 TCIU of wild-type HIV-1 JR-CSF or 3.6×10 5 TCIU of HIV JR-CSF Δ vif . (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 17 viral DNA sequences amplified from the tissues of infected mice. Viral DNA from mice infected with HIV JR-CSF Δ vif had 25%–65% of G G sites mutated. (D) G to A mutational profile of all viral DNA from mice infected with HIV JR-CSF Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites. D4-D8, WT2-WT3, NSG-hu mice. D9-D11, NSG-BLT mice.

    Article Snippet: Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche).

    Techniques: In Vivo, Mouse Assay, Infection, Nested PCR, Amplification, Mutagenesis

    Restoration of vif occurs only following direct virus injection into the thymus. (A) Longitudinal analysis of plasma viral load in NSG-BLT humanized mice infected with HIV JR-CSF vif FS directly into the human thymic implant, spleen, liver, or lung. Solid symbols represent mice infected with a low dose of virus (9×10 4 TCIU), open symbols represent the high dose infection (3.6×10 5 TCIU). (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 44 viral DNA sequences amplified from the tissues of mice injected with HIV JR-CSF vif FS. Mice injected into the thymic implant had no G G to A G mutations in any tissue whereas HIV DNA from four tissues of mouse FS24 injected into the spleen is hypermutated. (D) G to A mutational profile of all viral DNA from mouse FS24 which failed to restore the vif ORF. Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites.

    Journal: PLoS Pathogens

    Article Title: HIV Restriction by APOBEC3 in Humanized Mice

    doi: 10.1371/journal.ppat.1003242

    Figure Lengend Snippet: Restoration of vif occurs only following direct virus injection into the thymus. (A) Longitudinal analysis of plasma viral load in NSG-BLT humanized mice infected with HIV JR-CSF vif FS directly into the human thymic implant, spleen, liver, or lung. Solid symbols represent mice infected with a low dose of virus (9×10 4 TCIU), open symbols represent the high dose infection (3.6×10 5 TCIU). (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 44 viral DNA sequences amplified from the tissues of mice injected with HIV JR-CSF vif FS. Mice injected into the thymic implant had no G G to A G mutations in any tissue whereas HIV DNA from four tissues of mouse FS24 injected into the spleen is hypermutated. (D) G to A mutational profile of all viral DNA from mouse FS24 which failed to restore the vif ORF. Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites.

    Article Snippet: Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche).

    Techniques: Injection, Mouse Assay, Infection, Nested PCR, Amplification, Mutagenesis

    Human APOBEC3 exerts a strong selective pressure on HIV-1 JR-CSF containing a frameshift in vif . (A) Plasma viral load analysis in humanized mice intravenously infected with 9×10 4 or 3.6×10 5 TCIU of HIV JR-CSF vif FS. Viral RNA was not detected in the plasma (circles, n = 10) unless the vif ORF is restored (triangles, n = 6). The appearance of plasma viremia was delayed by 4 weeks in one of these mice. (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice intravenously infected with HIV JR-CSF vif FS. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Viral DNA is sparsely present in tissues from mice where vif was not restored (indicated as FS1–10). In contrast, all tissues analyzed from the six mice where vif was restored had viral DNA present (FS11–16). N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 76 viral DNA sequences amplified from the tissues of HIV JR-CSF vif FS infected mice where vif was not restored (40% of all G G sites mutated) or mice where vif was restored (no hypermutation). Data represent mean +/− SEM. (D) G to A mutational profile of all viral DNA from mice infected with HIV JR-CSF Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites. FS1–FS9, FS11–FS14, NSG-hu mice. FS10, FS15–FS16 NSG-BLT mice.

    Journal: PLoS Pathogens

    Article Title: HIV Restriction by APOBEC3 in Humanized Mice

    doi: 10.1371/journal.ppat.1003242

    Figure Lengend Snippet: Human APOBEC3 exerts a strong selective pressure on HIV-1 JR-CSF containing a frameshift in vif . (A) Plasma viral load analysis in humanized mice intravenously infected with 9×10 4 or 3.6×10 5 TCIU of HIV JR-CSF vif FS. Viral RNA was not detected in the plasma (circles, n = 10) unless the vif ORF is restored (triangles, n = 6). The appearance of plasma viremia was delayed by 4 weeks in one of these mice. (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice intravenously infected with HIV JR-CSF vif FS. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Viral DNA is sparsely present in tissues from mice where vif was not restored (indicated as FS1–10). In contrast, all tissues analyzed from the six mice where vif was restored had viral DNA present (FS11–16). N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites ( G G, G A, G Y) that were mutated in 76 viral DNA sequences amplified from the tissues of HIV JR-CSF vif FS infected mice where vif was not restored (40% of all G G sites mutated) or mice where vif was restored (no hypermutation). Data represent mean +/− SEM. (D) G to A mutational profile of all viral DNA from mice infected with HIV JR-CSF Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites. FS1–FS9, FS11–FS14, NSG-hu mice. FS10, FS15–FS16 NSG-BLT mice.

    Article Snippet: Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche).

    Techniques: Mouse Assay, Infection, Nested PCR, Amplification, Mutagenesis

    Human APOBEC3 rapidly restricts vif -deleted HIV-1 JR-CSF in vivo . (A) Replication of HIV JR-CSF , HIV JR-CSF Δ vif , and HIV JR-CSF vif FS in CEM-SS cells expressing CCR5 (CEM-SS CCR5). Culture supernatant was assayed for p24 Gag by ELISA at three day intervals to determine the replication kinetics of the mutant viruses. (B) Nested PCR amplification of viral DNA from the tissues obtained one week post-exposure from a representative NSG-hu mouse infected with 9×10 4 TCIU of wild-type HIV-1 JR-CSF (WT1) or from three mice infected with 3.6×10 5 TCIU of HIV JR-CSF Δ vif (indicated as D1–3). (C) Highlighter sequence analysis of 7 wild-type and 3 Δ vif HIV DNA sequences. Amplified viral DNA from panel A showed no APOBEC3 induced mutations in HIV JR-CSF (WT1 all sequence from tissues is shown together). In contrast, viral DNA from all positive tissues obtained from HIV JR-CSF Δ vif infected mice had G to A (green lines) and/or C to T mutations (red lines). HIV-1 JR-CSF nucleotide numbers are indicated at the bottom.

    Journal: PLoS Pathogens

    Article Title: HIV Restriction by APOBEC3 in Humanized Mice

    doi: 10.1371/journal.ppat.1003242

    Figure Lengend Snippet: Human APOBEC3 rapidly restricts vif -deleted HIV-1 JR-CSF in vivo . (A) Replication of HIV JR-CSF , HIV JR-CSF Δ vif , and HIV JR-CSF vif FS in CEM-SS cells expressing CCR5 (CEM-SS CCR5). Culture supernatant was assayed for p24 Gag by ELISA at three day intervals to determine the replication kinetics of the mutant viruses. (B) Nested PCR amplification of viral DNA from the tissues obtained one week post-exposure from a representative NSG-hu mouse infected with 9×10 4 TCIU of wild-type HIV-1 JR-CSF (WT1) or from three mice infected with 3.6×10 5 TCIU of HIV JR-CSF Δ vif (indicated as D1–3). (C) Highlighter sequence analysis of 7 wild-type and 3 Δ vif HIV DNA sequences. Amplified viral DNA from panel A showed no APOBEC3 induced mutations in HIV JR-CSF (WT1 all sequence from tissues is shown together). In contrast, viral DNA from all positive tissues obtained from HIV JR-CSF Δ vif infected mice had G to A (green lines) and/or C to T mutations (red lines). HIV-1 JR-CSF nucleotide numbers are indicated at the bottom.

    Article Snippet: Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche).

    Techniques: In Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Mutagenesis, Nested PCR, Amplification, Infection, Mouse Assay, Sequencing

    Sustained Vif-independent replication of CXCR4 tropic HIV-1. (A) Plasma viral load was monitored in NSG-BLT humanized mice infected with 3.6×10 5 TCIU HIV LAI Δ vif directly into the human thymic implant, spleen, liver, or lung. Direct injection of HIV LAI Δ vif into the thymus resulted in plasma viremia in 4/4 infections. (B) Longitudinal analysis of plasma viral load in humanized mice infected intravenously with 3.6×10 5 TCIU of HIV LAI Δ vif (n = 7) or 3–9×10 4 TCIU of wild-type HIV LAI (n = 6). Data represent mean +/− SEM. (C) Longitudinal analysis of the percentage of CD4 + T cells in the peripheral blood of humanized mice infected in panel B. (D) Comparison of the G to A mutation frequency in the viral RNA from the plasma and the viral DNA from peripheral blood cells from mice intravenously infected with HIV LAI Δ vif . Data represent mean +/− SEM from 18 sequences, ** p = 0.0066. (E) Detection of HIV DNA (+) by nested PCR from the tissues of BLT humanized mice in panels A and B. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Direct injection of HIV LAI Δ vif into the liver, lung or spleen resulted in limited tissue distribution of viral DNA. (F) Comparison of the G to A mutation frequency in viral DNA from the thymus compared to viral DNA from other tissues of mice infected intrathymically (n = 4) and intravenously (n = 7) with HIV LAI Δ vif . Data represent mean +/− SEM from 83 sequences. (G) G to A mutational profile of all viral DNA from mice infected with HIV LAI Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites.

    Journal: PLoS Pathogens

    Article Title: HIV Restriction by APOBEC3 in Humanized Mice

    doi: 10.1371/journal.ppat.1003242

    Figure Lengend Snippet: Sustained Vif-independent replication of CXCR4 tropic HIV-1. (A) Plasma viral load was monitored in NSG-BLT humanized mice infected with 3.6×10 5 TCIU HIV LAI Δ vif directly into the human thymic implant, spleen, liver, or lung. Direct injection of HIV LAI Δ vif into the thymus resulted in plasma viremia in 4/4 infections. (B) Longitudinal analysis of plasma viral load in humanized mice infected intravenously with 3.6×10 5 TCIU of HIV LAI Δ vif (n = 7) or 3–9×10 4 TCIU of wild-type HIV LAI (n = 6). Data represent mean +/− SEM. (C) Longitudinal analysis of the percentage of CD4 + T cells in the peripheral blood of humanized mice infected in panel B. (D) Comparison of the G to A mutation frequency in the viral RNA from the plasma and the viral DNA from peripheral blood cells from mice intravenously infected with HIV LAI Δ vif . Data represent mean +/− SEM from 18 sequences, ** p = 0.0066. (E) Detection of HIV DNA (+) by nested PCR from the tissues of BLT humanized mice in panels A and B. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Direct injection of HIV LAI Δ vif into the liver, lung or spleen resulted in limited tissue distribution of viral DNA. (F) Comparison of the G to A mutation frequency in viral DNA from the thymus compared to viral DNA from other tissues of mice infected intrathymically (n = 4) and intravenously (n = 7) with HIV LAI Δ vif . Data represent mean +/− SEM from 83 sequences. (G) G to A mutational profile of all viral DNA from mice infected with HIV LAI Δ vif . Percentages indicate the proportion of G to A mutations occurring at G G (blue), G A (red), or G Y (black) sites.

    Article Snippet: Viral DNA or cDNA was amplified by nested PCR using the Expand High Fidelity PCR System (Roche).

    Techniques: Mouse Assay, Infection, Injection, Mutagenesis, Nested PCR, Amplification