Structured Review

Roche genomic dna
Generation and validation of a Casp2 C320S mice. ( a ) A representative <t>DNA</t> agarose gel image from <t>genotyping</t> of WT, Casp2 +/C320S (heterozygous) and Casp2 C320S/C320S (homozygous) mice. The size of expected DNA fragments generated per genotype are indicated (bp). ( b ) DNA sequencing confirmation of the different genotypes as indicated. The red and blue arrows indicate the point mutation sites (G, G/C or C) and the silent mutation site (T, T/A or A), respectively. ( c ) Immunoblot analysis of caspase-2 expression in splenocytes from WT and Casp2 C320S/C320S mice. β-actin is shown as loading control. ( d ) Genotype frequencies of the progeny from Casp2 +/C320S (heterozygous) intercrossing including observed and expected numbers of offspring. Observed and expected frequency (%) is indicated in parentheses. Statistical comparison was performed by χ 2 test.
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1) Product Images from "Caspase-2-mediated cell death is required for deleting aneuploid cells"

Article Title: Caspase-2-mediated cell death is required for deleting aneuploid cells

Journal: Oncogene

doi: 10.1038/onc.2016.423

Generation and validation of a Casp2 C320S mice. ( a ) A representative DNA agarose gel image from genotyping of WT, Casp2 +/C320S (heterozygous) and Casp2 C320S/C320S (homozygous) mice. The size of expected DNA fragments generated per genotype are indicated (bp). ( b ) DNA sequencing confirmation of the different genotypes as indicated. The red and blue arrows indicate the point mutation sites (G, G/C or C) and the silent mutation site (T, T/A or A), respectively. ( c ) Immunoblot analysis of caspase-2 expression in splenocytes from WT and Casp2 C320S/C320S mice. β-actin is shown as loading control. ( d ) Genotype frequencies of the progeny from Casp2 +/C320S (heterozygous) intercrossing including observed and expected numbers of offspring. Observed and expected frequency (%) is indicated in parentheses. Statistical comparison was performed by χ 2 test.
Figure Legend Snippet: Generation and validation of a Casp2 C320S mice. ( a ) A representative DNA agarose gel image from genotyping of WT, Casp2 +/C320S (heterozygous) and Casp2 C320S/C320S (homozygous) mice. The size of expected DNA fragments generated per genotype are indicated (bp). ( b ) DNA sequencing confirmation of the different genotypes as indicated. The red and blue arrows indicate the point mutation sites (G, G/C or C) and the silent mutation site (T, T/A or A), respectively. ( c ) Immunoblot analysis of caspase-2 expression in splenocytes from WT and Casp2 C320S/C320S mice. β-actin is shown as loading control. ( d ) Genotype frequencies of the progeny from Casp2 +/C320S (heterozygous) intercrossing including observed and expected numbers of offspring. Observed and expected frequency (%) is indicated in parentheses. Statistical comparison was performed by χ 2 test.

Techniques Used: Mouse Assay, Agarose Gel Electrophoresis, Generated, DNA Sequencing, Mutagenesis, Expressing

2) Product Images from "DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO"

Article Title: DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO

Journal: Molecular Systems Biology

doi: 10.1038/msb.2010.54

The DAM identification (DamID) procedure in C. elegans . ( A ) How DamID works. A fusion protein consisting of DNA adenine methyltransferase (DAM) and the protein of interest methylates GATC sites near binding sites. Genomic DNA is digested with Dpn I, which cuts only methylated GATC sites. Adaptors are added, and DNA is digested with Dpn II (which cuts at unmethylated GATC sites) to assure selective amplification of methylated DNA. A parallel DAM-only experiment is also performed to control for non-specific methylation. Samples are then labeled and hybridized to arrays. ( B ) Schematic of plasmid constructs used for preparation of transgenic strains. ( C , D ) Transgene expression. Nuclear localization of GFP was detected in UL1782 animals (expressing DAM∷DAF-16∷GFP) (marked with arrows in C) in body wall muscle and anterior bulb of pharynx (circled) following heat shock. UL1787 animals (expressing DAM∷GFP) (D) do not show nuclear localization. ( E ) DAF-16∷DAM methylation profile for ist-1 , one of several evolutionarily conserved FoxO targets identified. ( F ) Average distribution of methylation (DAF-16∷DAM versus DAM) from peak center for 1135 peaks identified.
Figure Legend Snippet: The DAM identification (DamID) procedure in C. elegans . ( A ) How DamID works. A fusion protein consisting of DNA adenine methyltransferase (DAM) and the protein of interest methylates GATC sites near binding sites. Genomic DNA is digested with Dpn I, which cuts only methylated GATC sites. Adaptors are added, and DNA is digested with Dpn II (which cuts at unmethylated GATC sites) to assure selective amplification of methylated DNA. A parallel DAM-only experiment is also performed to control for non-specific methylation. Samples are then labeled and hybridized to arrays. ( B ) Schematic of plasmid constructs used for preparation of transgenic strains. ( C , D ) Transgene expression. Nuclear localization of GFP was detected in UL1782 animals (expressing DAM∷DAF-16∷GFP) (marked with arrows in C) in body wall muscle and anterior bulb of pharynx (circled) following heat shock. UL1787 animals (expressing DAM∷GFP) (D) do not show nuclear localization. ( E ) DAF-16∷DAM methylation profile for ist-1 , one of several evolutionarily conserved FoxO targets identified. ( F ) Average distribution of methylation (DAF-16∷DAM versus DAM) from peak center for 1135 peaks identified.

Techniques Used: Binding Assay, Methylation, Amplification, Labeling, Plasmid Preparation, Construct, Transgenic Assay, Expressing

3) Product Images from "Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization"

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065542

GRN played a pivotal role in the ALD-DNA-induced M2b polarization in vitro . (A–B) Primary macrophages were stimulated with increasing amounts of ALD-DNA for 24 h. mRNA levels of GRN in macrophages were analyzed by real time PCR analysis (A), and protein levels of GRN in the supernatants of macrophages were analyzed by western blot (B). Above, quantitative results of western blots, the band intensity was measured by Image J; Below, representative western blots. Similar results were obtained in three independent experiments. Data are representative of results obtained in three independent experiments. Primary peritoneal macrophages were stimulated by ALD-DNA (50 µg/mL) with GRN (5 µg/mL) for 24 h (C and F), or were pretreated with elastase inhibitor (100 µM) or DMSO (0.1%) for 12 h (D and G), and then were exposed to ALD-DNA, UnALD-DNA, or PBS for another 24 h. Macrophages were transfected with control siRNA (200 nM) or GRN siRNA (siGRN, 200 nM). 36 h posttransfection, macrophages were stimulated with PBS, UnALD-DNA or ALD-DNA (50 µg/mL) (E and H). (C–E) ELISA assay was used to analyze the levels of TNF-α, IL-1β, IL-6, IL-10, IL-12, and MCP-1 in the culture supernatants of macrophages. Data are means ± SD of three independent experiments. (F–H) Western blot analysis was used to analyze the protein levels of iNOS in macrophages. Data are representative of three separate experiments. Similar results were obtained in three independent experiments. Band intensity was measured by Image J and the ratios of iNOS to β-actin were calculated. *, P
Figure Legend Snippet: GRN played a pivotal role in the ALD-DNA-induced M2b polarization in vitro . (A–B) Primary macrophages were stimulated with increasing amounts of ALD-DNA for 24 h. mRNA levels of GRN in macrophages were analyzed by real time PCR analysis (A), and protein levels of GRN in the supernatants of macrophages were analyzed by western blot (B). Above, quantitative results of western blots, the band intensity was measured by Image J; Below, representative western blots. Similar results were obtained in three independent experiments. Data are representative of results obtained in three independent experiments. Primary peritoneal macrophages were stimulated by ALD-DNA (50 µg/mL) with GRN (5 µg/mL) for 24 h (C and F), or were pretreated with elastase inhibitor (100 µM) or DMSO (0.1%) for 12 h (D and G), and then were exposed to ALD-DNA, UnALD-DNA, or PBS for another 24 h. Macrophages were transfected with control siRNA (200 nM) or GRN siRNA (siGRN, 200 nM). 36 h posttransfection, macrophages were stimulated with PBS, UnALD-DNA or ALD-DNA (50 µg/mL) (E and H). (C–E) ELISA assay was used to analyze the levels of TNF-α, IL-1β, IL-6, IL-10, IL-12, and MCP-1 in the culture supernatants of macrophages. Data are means ± SD of three independent experiments. (F–H) Western blot analysis was used to analyze the protein levels of iNOS in macrophages. Data are representative of three separate experiments. Similar results were obtained in three independent experiments. Band intensity was measured by Image J and the ratios of iNOS to β-actin were calculated. *, P

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

GRN aggravated lupus nephritis in lupus model by enhancing ALD-DNA-induced M2b polarization. CD11b + /F4/80 high renal macrophages were sorted from nephritic single-cell suspensions from pGRN-treated, pcDNA3.1-treated, or LV-shGRN-injected, LV-shNC-injected lupus model by flow cytometry. (A) mRNA levels of TNF-α , IL-1β , IL-6 , IL-10 , IL-12 , MCP-1 , Nos2 (iNOS) and Agr1 in the renal macrophages screened from pGRN or pcDNA3.1-treated lupus mice were evaluated by real-time PCR. Data are means ± SD from 8 mice in each group. (B) Real time analysis for the mRNA levels of TNF-α , IL-1β , IL-6 , IL-10 , IL-12 , MCP-1 , Nos2 (iNOS) and Agr1 in renal macrophages from LV-shGRN-injected lupus mice and control mice. Data are means ± SD from 8 mice in each group. *, P
Figure Legend Snippet: GRN aggravated lupus nephritis in lupus model by enhancing ALD-DNA-induced M2b polarization. CD11b + /F4/80 high renal macrophages were sorted from nephritic single-cell suspensions from pGRN-treated, pcDNA3.1-treated, or LV-shGRN-injected, LV-shNC-injected lupus model by flow cytometry. (A) mRNA levels of TNF-α , IL-1β , IL-6 , IL-10 , IL-12 , MCP-1 , Nos2 (iNOS) and Agr1 in the renal macrophages screened from pGRN or pcDNA3.1-treated lupus mice were evaluated by real-time PCR. Data are means ± SD from 8 mice in each group. (B) Real time analysis for the mRNA levels of TNF-α , IL-1β , IL-6 , IL-10 , IL-12 , MCP-1 , Nos2 (iNOS) and Agr1 in renal macrophages from LV-shGRN-injected lupus mice and control mice. Data are means ± SD from 8 mice in each group. *, P

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

4) Product Images from "CpG island methylation is a common finding in colorectal cancer cell lines"

Article Title: CpG island methylation is a common finding in colorectal cancer cell lines

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6600699

Analysis of methylation of p16 and MINT loci in colorectal cell lines. ( A ) For p16 promoter analysis, bisulphite-modified cell line DNA was amplified in separate reactions using primers specific for unmethylated (U) or methylated (M) template (methylation-specific PCR - MSP). Examples of methylated cell lines are T84 and RKO; these show amplification in the methylated reaction only. Presence of PCR product in both unmethylated and methylated reactions of HCT116 is indicative of partial methylation at p16 . LIM1215 is shown as an example of a cell line unmethylated at p16 . ( B ) For analysis of methylation at MINT loci, bisulphite-modified DNA was first PCR amplified using appropriate loci-specific primers, and then the PCR products were digested with restriction enzymes that only cut amplicons generated from the methylated template. MINT2 is shown as a representative MINT locus. Resistance to digestion indicates an unmethylated template. Shown are cell lines with partial methylation at MINT 2, CACO2 and RKO, a fully methylated cell line, COLO205, and an unmethylated cell line LIM1215. Undigested amplicons are shown in the last lane. Molecular weight (MW) marker in A and B is pUC19/ Msp I.
Figure Legend Snippet: Analysis of methylation of p16 and MINT loci in colorectal cell lines. ( A ) For p16 promoter analysis, bisulphite-modified cell line DNA was amplified in separate reactions using primers specific for unmethylated (U) or methylated (M) template (methylation-specific PCR - MSP). Examples of methylated cell lines are T84 and RKO; these show amplification in the methylated reaction only. Presence of PCR product in both unmethylated and methylated reactions of HCT116 is indicative of partial methylation at p16 . LIM1215 is shown as an example of a cell line unmethylated at p16 . ( B ) For analysis of methylation at MINT loci, bisulphite-modified DNA was first PCR amplified using appropriate loci-specific primers, and then the PCR products were digested with restriction enzymes that only cut amplicons generated from the methylated template. MINT2 is shown as a representative MINT locus. Resistance to digestion indicates an unmethylated template. Shown are cell lines with partial methylation at MINT 2, CACO2 and RKO, a fully methylated cell line, COLO205, and an unmethylated cell line LIM1215. Undigested amplicons are shown in the last lane. Molecular weight (MW) marker in A and B is pUC19/ Msp I.

Techniques Used: Methylation, Modification, Amplification, Polymerase Chain Reaction, Generated, Molecular Weight, Marker

Analysis of hMLH1 methylation and expression in colorectal cell lines. ( A ) Both the A region (top panel) and C region (bottom panel) of the hMLH1 promoter were amplified from bisulphite-treated DNA. Bst U1 restriction enzyme digest was performed to distinguish amplicons from methylated or unmethylated template; BstU1 will cut only amplicons generated from methylated template. Undigested PCR product is shown in the control lane (-). Lane 1 shows an example of a cell line unmethylated at both the A and C regions, DLD1. Lane 2 shows SW403 that is methylated at the A region, but not C. Lane 3 shows LS411 that is partially methylated at both regions and Lane 4 shows a fully methylated cell line, RKO. Peripheral blood (PB) DNA used as a control is always unmethylated at both the A and C regions. MW market is pUC19/ Msp I. ( B ) Representative Western blot analysis of hMLH1 expression in colorectal cell lines with various combinations of A and C region methylations. SW480 has no methylation at either region and has wild-type hMLH1 . HCT116 also is unmethylated at both regions; however, this cell line has a frameshift mutation in one allele of hMLH1 , leading to a reduction in expression levels. Cell lines exhibiting either full (RKO) or partial (LS411) methylation at both the A and C regions show a loss of hMLH1 protein.
Figure Legend Snippet: Analysis of hMLH1 methylation and expression in colorectal cell lines. ( A ) Both the A region (top panel) and C region (bottom panel) of the hMLH1 promoter were amplified from bisulphite-treated DNA. Bst U1 restriction enzyme digest was performed to distinguish amplicons from methylated or unmethylated template; BstU1 will cut only amplicons generated from methylated template. Undigested PCR product is shown in the control lane (-). Lane 1 shows an example of a cell line unmethylated at both the A and C regions, DLD1. Lane 2 shows SW403 that is methylated at the A region, but not C. Lane 3 shows LS411 that is partially methylated at both regions and Lane 4 shows a fully methylated cell line, RKO. Peripheral blood (PB) DNA used as a control is always unmethylated at both the A and C regions. MW market is pUC19/ Msp I. ( B ) Representative Western blot analysis of hMLH1 expression in colorectal cell lines with various combinations of A and C region methylations. SW480 has no methylation at either region and has wild-type hMLH1 . HCT116 also is unmethylated at both regions; however, this cell line has a frameshift mutation in one allele of hMLH1 , leading to a reduction in expression levels. Cell lines exhibiting either full (RKO) or partial (LS411) methylation at both the A and C regions show a loss of hMLH1 protein.

Techniques Used: Methylation, Expressing, Amplification, Generated, Polymerase Chain Reaction, Western Blot, Mutagenesis

5) Product Images from "Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides 1 1 [W] 1 [W] [OPEN]"

Article Title: Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides 1 1 [W] 1 [W] [OPEN]

Journal: Plant Physiology

doi: 10.1104/pp.113.224675

Southern-blot analysis of T1 sporophytes of P. vittata (A, B, and C) and C. thalictroides (D). Genomic DNA from fronds were digested with Cla -labeled hpt gene fragment. Lanes 2 to 5, 7 to 10, and 12 to 22: transgenic P. vittata.
Figure Legend Snippet: Southern-blot analysis of T1 sporophytes of P. vittata (A, B, and C) and C. thalictroides (D). Genomic DNA from fronds were digested with Cla -labeled hpt gene fragment. Lanes 2 to 5, 7 to 10, and 12 to 22: transgenic P. vittata.

Techniques Used: Southern Blot, Labeling, Transgenic Assay

6) 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

7) Product Images from "Characterization of morphology and resistance to Blumeria graminis of winter triticale monosomic addition lines with chromosome 2D of Aegilopstauschii"

Article Title: Characterization of morphology and resistance to Blumeria graminis of winter triticale monosomic addition lines with chromosome 2D of Aegilopstauschii

Journal: Plant Cell Reports

doi: 10.1007/s00299-016-2023-x

Idiogram and karyotype of two types of additional chromosomes of Ae . tauschii detected in the background of BC 2 F 5 and BC 2 F 6 hybrids of triticale showing genomic distribution of repetitive DNA sequence pAs1 ( red ). a Normal chromosome 2D, b chromosome 2D with rearrangement (color figure online)
Figure Legend Snippet: Idiogram and karyotype of two types of additional chromosomes of Ae . tauschii detected in the background of BC 2 F 5 and BC 2 F 6 hybrids of triticale showing genomic distribution of repetitive DNA sequence pAs1 ( red ). a Normal chromosome 2D, b chromosome 2D with rearrangement (color figure online)

Techniques Used: Sequencing

Mitotic chromosomes of BC 2 F 5 ( Ae . tauschii × S . cereale ) × Triticosecale cv. Bogo hybrid analyzed using a FISH with 5S rDNA ( red ) and 35S rDNA ( green ) probes, b FISH with pAs1 ( red ) and pSc119.2 ( green ) probes, c GISH with total genomic DNA probes of Ae . tauschii (D, green ) and triticale (ABR, orange ), d multicolor GISH with total genomic DNA probes of T . monococcum (A, green ), S . cereale and Ae . speltoides (R and B, blue ), and Ae . tauschii (D, red ). Arrows indicate the introgressed chromosome 2D. Scale bar 10 µm (color figure online)
Figure Legend Snippet: Mitotic chromosomes of BC 2 F 5 ( Ae . tauschii × S . cereale ) × Triticosecale cv. Bogo hybrid analyzed using a FISH with 5S rDNA ( red ) and 35S rDNA ( green ) probes, b FISH with pAs1 ( red ) and pSc119.2 ( green ) probes, c GISH with total genomic DNA probes of Ae . tauschii (D, green ) and triticale (ABR, orange ), d multicolor GISH with total genomic DNA probes of T . monococcum (A, green ), S . cereale and Ae . speltoides (R and B, blue ), and Ae . tauschii (D, red ). Arrows indicate the introgressed chromosome 2D. Scale bar 10 µm (color figure online)

Techniques Used: Fluorescence In Situ Hybridization

8) Product Images from "Development of a Highly Sensitive and Specific Method for Detection of Circulating Tumor Cells Harboring Somatic Mutations in Non-Small-Cell Lung Cancer Patients"

Article Title: Development of a Highly Sensitive and Specific Method for Detection of Circulating Tumor Cells Harboring Somatic Mutations in Non-Small-Cell Lung Cancer Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085350

Sensitivity of EGFR DelEx19 mutation detection by real-time polymerase chain reaction and melting curve analysis. A) EGFR DelEx19 mutation detection in serially diluted DNA (50 ng/reaction) from A431 cells ( EGFR -wild type control) and NCI-HCC-827 cells ( EGFR DelEx19 mutant control 1). Melting peaks indicative of EGFR -wild type DNA (right) and EGFR DelEx19 (left) can be clearly distinguished. Real-time PCR reactions were carried out without addition of locked nucleic acids (LNA) and in serial DNA dilutions of up to 1∶16. Water (H 2 O, bottom line) and and 50 ng of undiluted genomic DNA EGFR -wild type (A431) and EGFR -mutant cells (NCI-HCC-827) were included as controls (representative examples of duplicate reactions). B) Reactions were conducted as in (A) but with addition of LNA (6 pmol). Note suppression of the EGFR -wild type signal, which allowed discrimination of the EGFR DelEx19 mutation signal up to a dilution of 1∶1,024 (
Figure Legend Snippet: Sensitivity of EGFR DelEx19 mutation detection by real-time polymerase chain reaction and melting curve analysis. A) EGFR DelEx19 mutation detection in serially diluted DNA (50 ng/reaction) from A431 cells ( EGFR -wild type control) and NCI-HCC-827 cells ( EGFR DelEx19 mutant control 1). Melting peaks indicative of EGFR -wild type DNA (right) and EGFR DelEx19 (left) can be clearly distinguished. Real-time PCR reactions were carried out without addition of locked nucleic acids (LNA) and in serial DNA dilutions of up to 1∶16. Water (H 2 O, bottom line) and and 50 ng of undiluted genomic DNA EGFR -wild type (A431) and EGFR -mutant cells (NCI-HCC-827) were included as controls (representative examples of duplicate reactions). B) Reactions were conducted as in (A) but with addition of LNA (6 pmol). Note suppression of the EGFR -wild type signal, which allowed discrimination of the EGFR DelEx19 mutation signal up to a dilution of 1∶1,024 (

Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction

9) Product Images from "NKL homeobox gene NKX2-2 is aberrantly expressed in Hodgkin lymphoma"

Article Title: NKL homeobox gene NKX2-2 is aberrantly expressed in Hodgkin lymphoma

Journal: Oncotarget

doi: 10.18632/oncotarget.26459

DAZAP2 and SMURF2 are deregulated modulators of IL17RB activity (A) RQ-PCR analysis of DAZAP2 in HL cell lines (left). RQ-PCR analysis of DAZAP2, FOXG1 and NKX2-2 in DEV cells treated for siRNA-mediated knockdown of DAZAP2 (right). (B) Genomic profiling data for chromosome 12 of HL cell line DEV. The locus of DAZAP2 is indicated and located in a region of copy number gain. (C) RQ-PCR analysis of SMURF2 in HL cell lines (left) and in primary hematopoietic cell/tissue samples (right). (D) Genomic profiling data for chromosome 17 of HL cell line DEV. The locus of SMURF2 is indicated and located in deleted region. Enlargement of this deleted region demonstrates that the promoter region and the 5’-primed end of SMURF2 is lost (below). (E) Quantification of exon 1 of SMURF2 in genomic DNA of selected HL cell lines by Q-PCR. The gene MEF2C served as reference. (F) RQ-PCR analysis of NKX2-2 in DEV cells treated with forced expression of SMURF2.
Figure Legend Snippet: DAZAP2 and SMURF2 are deregulated modulators of IL17RB activity (A) RQ-PCR analysis of DAZAP2 in HL cell lines (left). RQ-PCR analysis of DAZAP2, FOXG1 and NKX2-2 in DEV cells treated for siRNA-mediated knockdown of DAZAP2 (right). (B) Genomic profiling data for chromosome 12 of HL cell line DEV. The locus of DAZAP2 is indicated and located in a region of copy number gain. (C) RQ-PCR analysis of SMURF2 in HL cell lines (left) and in primary hematopoietic cell/tissue samples (right). (D) Genomic profiling data for chromosome 17 of HL cell line DEV. The locus of SMURF2 is indicated and located in deleted region. Enlargement of this deleted region demonstrates that the promoter region and the 5’-primed end of SMURF2 is lost (below). (E) Quantification of exon 1 of SMURF2 in genomic DNA of selected HL cell lines by Q-PCR. The gene MEF2C served as reference. (F) RQ-PCR analysis of NKX2-2 in DEV cells treated with forced expression of SMURF2.

Techniques Used: Activity Assay, Polymerase Chain Reaction, Expressing

10) Product Images from "ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development"

Article Title: ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201403868

ZIPCO-F parasites develop normally in blood stages but sporozoites are less infectious to the mammalian host Schematic representation of P. berghei zipco targeted gene disruption. The plasmid pBC- zipco -hDHFR used for homologous recombination is shown on the top with the hDHFR cassette indicated by a black arrow. The endogenous locus (Locus -WT ) is shown in the middle; the dotted rectangle indicates the region deleted and replaced by the hDHFR cassette. The resulting recombinant locus is shown in the lower part. Sizes of restriction fragments obtained after digestion of genomic DNA with Bsm I and Hinc II are indicated by double-sided arrows. The probe used for Southern blot analysis (see panel 2B) is shown as a black bar. Analysis of zipco locus in WT and recombinant parasites. Genomic DNAs from NK65 (WT), ANKA-GFP (WT-F), and recombinant clones ZIPCO and ZIPCO-F were digested with Bsm I and Hinc II. A probe from the 3′UTR region (shown in 2A) was used for analysis. The WT (1,348 bp) and recombinant (2,480 bp) restriction fragments detected are indicated by an arrowhead and arrow, respectively. The 949 bp Hinc II fragment, present in both WT and recombinant loci, is indicated by a small arrowhead. Graph showing the multiplication of WT-F and ZIPCO-F blood stages. Mice were injected with mixed blood stages to obtain an initial parasitemia of 0.001%. The parasitemia was followed daily for 4 days on Geimsa-stained blood smears. Graph represents the mean parasitaemia ± SD ( n = 6 mice in each group). Graph showing the mean parasitemia ± SD of mice after intradermal injection with 25,000 WT-F or ZIPCO-F sporozoites. ( n = 5 mice in each group). Graph showing the mean parasitemia ± SD of mice after intravenous injection with 25,000 WT-F or ZIPCO-F sporozoites. ( n = 5 mice in each group). Source data are available online for this figure.
Figure Legend Snippet: ZIPCO-F parasites develop normally in blood stages but sporozoites are less infectious to the mammalian host Schematic representation of P. berghei zipco targeted gene disruption. The plasmid pBC- zipco -hDHFR used for homologous recombination is shown on the top with the hDHFR cassette indicated by a black arrow. The endogenous locus (Locus -WT ) is shown in the middle; the dotted rectangle indicates the region deleted and replaced by the hDHFR cassette. The resulting recombinant locus is shown in the lower part. Sizes of restriction fragments obtained after digestion of genomic DNA with Bsm I and Hinc II are indicated by double-sided arrows. The probe used for Southern blot analysis (see panel 2B) is shown as a black bar. Analysis of zipco locus in WT and recombinant parasites. Genomic DNAs from NK65 (WT), ANKA-GFP (WT-F), and recombinant clones ZIPCO and ZIPCO-F were digested with Bsm I and Hinc II. A probe from the 3′UTR region (shown in 2A) was used for analysis. The WT (1,348 bp) and recombinant (2,480 bp) restriction fragments detected are indicated by an arrowhead and arrow, respectively. The 949 bp Hinc II fragment, present in both WT and recombinant loci, is indicated by a small arrowhead. Graph showing the multiplication of WT-F and ZIPCO-F blood stages. Mice were injected with mixed blood stages to obtain an initial parasitemia of 0.001%. The parasitemia was followed daily for 4 days on Geimsa-stained blood smears. Graph represents the mean parasitaemia ± SD ( n = 6 mice in each group). Graph showing the mean parasitemia ± SD of mice after intradermal injection with 25,000 WT-F or ZIPCO-F sporozoites. ( n = 5 mice in each group). Graph showing the mean parasitemia ± SD of mice after intravenous injection with 25,000 WT-F or ZIPCO-F sporozoites. ( n = 5 mice in each group). Source data are available online for this figure.

Techniques Used: Plasmid Preparation, Homologous Recombination, Recombinant, Southern Blot, Clone Assay, Mouse Assay, Injection, Staining

11) Product Images from "Characterization of a Novel Type II Restriction-Modification System, Sth368I, Encoded by the Integrative Element ICESt1 of Streptococcus thermophilus CNRZ368"

Article Title: Characterization of a Novel Type II Restriction-Modification System, Sth368I, Encoded by the Integrative Element ICESt1 of Streptococcus thermophilus CNRZ368

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.67.4.1522-1528.2001

Electrophoresis of Sau 3AI digestion assays of DNA fragments overlapping ICE St1 . Lane 1, λ DNA digested by Pst I; lane 2, λNST101; lane 3, λNST106; lane 4, λNST107; lane 5, λNST108; lane 6, λNST113; lane 7, pNST144; lane 8, λ DNA digested by Hind III.
Figure Legend Snippet: Electrophoresis of Sau 3AI digestion assays of DNA fragments overlapping ICE St1 . Lane 1, λ DNA digested by Pst I; lane 2, λNST101; lane 3, λNST106; lane 4, λNST107; lane 5, λNST108; lane 6, λNST113; lane 7, pNST144; lane 8, λ DNA digested by Hind III.

Techniques Used: Electrophoresis

12) Product Images from "Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter"

Article Title: Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter

Journal: Journal of Clinical Investigation

doi:

PCR of human genomic DNA (lanes 1, 2) and BAC clones 452D14 (lanes 3–6) and 305N23 (lanes 7–10). Lanes 1, 3, 4, 7, and 8 are PCR products obtained with hUAT -specific primers. Lanes 2, 5, 6, 9, and 10 are PCR products obtained with hUAT2 -specific primers. Lane 0 is a 1-kbp DNA ladder.
Figure Legend Snippet: PCR of human genomic DNA (lanes 1, 2) and BAC clones 452D14 (lanes 3–6) and 305N23 (lanes 7–10). Lanes 1, 3, 4, 7, and 8 are PCR products obtained with hUAT -specific primers. Lanes 2, 5, 6, 9, and 10 are PCR products obtained with hUAT2 -specific primers. Lane 0 is a 1-kbp DNA ladder.

Techniques Used: Polymerase Chain Reaction, BAC Assay, Clone Assay

13) Product Images from "Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification"

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1016/j.omtm.2017.01.005

Evaluation of TALEN and CRISPR/Cas9 Activity (A) Positions of TALEN and CRISPR/Cas9 gRNA binding sites at the TRAC target site. (B) TALEN and CRISPR/Cas9 activity in the TRAC locus in K562 cells. (C) Activity of obligate heterodimeric TALENs in the TRAC locus in 293T cells. (D) TALEN and gRNA binding sites at TRBC1 and TRBC2 target locus. (E) TALEN activity in the TRBC1 and TRBC2 locus in 293T cells. (F) CRISPR/Cas9 activity in the TRBC1 and TRBC2 locus in K562 cells. (B, C, E, and F) PCR amplification of the target regions in the TCR loci produces upper bands. T7EI-mediated cleavage of NHEJ-originated heteroduplex DNA results in additional cleavage bands, marked by arrowheads. A SNP in the TRBC2 locus results in additional bands, marked by arrows ( > ). Ctrl, negative control; M, marker; Sp., length of spacer between TALEN binding sites in base pairs; TALEN G , GoldyTALEN; TALEN P(OH) , pTAL3 (obligate heterodimeric FokI domain); TALEN S , CAG-T7-TALEN(Sangamo)-Destination.
Figure Legend Snippet: Evaluation of TALEN and CRISPR/Cas9 Activity (A) Positions of TALEN and CRISPR/Cas9 gRNA binding sites at the TRAC target site. (B) TALEN and CRISPR/Cas9 activity in the TRAC locus in K562 cells. (C) Activity of obligate heterodimeric TALENs in the TRAC locus in 293T cells. (D) TALEN and gRNA binding sites at TRBC1 and TRBC2 target locus. (E) TALEN activity in the TRBC1 and TRBC2 locus in 293T cells. (F) CRISPR/Cas9 activity in the TRBC1 and TRBC2 locus in K562 cells. (B, C, E, and F) PCR amplification of the target regions in the TCR loci produces upper bands. T7EI-mediated cleavage of NHEJ-originated heteroduplex DNA results in additional cleavage bands, marked by arrowheads. A SNP in the TRBC2 locus results in additional bands, marked by arrows ( > ). Ctrl, negative control; M, marker; Sp., length of spacer between TALEN binding sites in base pairs; TALEN G , GoldyTALEN; TALEN P(OH) , pTAL3 (obligate heterodimeric FokI domain); TALEN S , CAG-T7-TALEN(Sangamo)-Destination.

Techniques Used: CRISPR, Activity Assay, Binding Assay, TALENs, Polymerase Chain Reaction, Amplification, Non-Homologous End Joining, Negative Control, Marker

14) Product Images from "Glycoprotein Ibα Kozak polymorphism in patients presenting with early-onset acute coronary syndrome"

Article Title: Glycoprotein Ibα Kozak polymorphism in patients presenting with early-onset acute coronary syndrome

Journal: Archives of Medical Science : AMS

doi: 10.5114/aoms.2016.63278

Assessment of GPIbα Kozak polymorphism. Individuals were genotyped by cleavage of PCR products with restriction endonuclease enzyme Psp5II. 1 and 5: 100 bp DNA marker, 2–4: The enzyme cleavage products of TT genotyped individuals (175, 157, 125 bp)
Figure Legend Snippet: Assessment of GPIbα Kozak polymorphism. Individuals were genotyped by cleavage of PCR products with restriction endonuclease enzyme Psp5II. 1 and 5: 100 bp DNA marker, 2–4: The enzyme cleavage products of TT genotyped individuals (175, 157, 125 bp)

Techniques Used: Polymerase Chain Reaction, Marker

15) Product Images from "Ras-association domain family 1C protein promotes breast cancer cell migration and attenuates apoptosis"

Article Title: Ras-association domain family 1C protein promotes breast cancer cell migration and attenuates apoptosis

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-562

T47D, MDA-MB231, and Hs578T cells stably transduced with an empty MLV-backbone (T47D-, MDA-, and Hs578T-BB) or MLV-HA-RASSF1C (T47D-, MDA-, and Hs578T-1C) vectors were treated with 1 μg/ml doxycycline for 14 days . Genomic DNA was isolated for DNA fragmentation analysis using Apoptotic DNA ladder (ADL) kit. It is clear that DNA fragmentation did not occur in all the three cell lines tested suggesting that RASSF1C over-expression does not induce apoptosis.
Figure Legend Snippet: T47D, MDA-MB231, and Hs578T cells stably transduced with an empty MLV-backbone (T47D-, MDA-, and Hs578T-BB) or MLV-HA-RASSF1C (T47D-, MDA-, and Hs578T-1C) vectors were treated with 1 μg/ml doxycycline for 14 days . Genomic DNA was isolated for DNA fragmentation analysis using Apoptotic DNA ladder (ADL) kit. It is clear that DNA fragmentation did not occur in all the three cell lines tested suggesting that RASSF1C over-expression does not induce apoptosis.

Techniques Used: Multiple Displacement Amplification, Stable Transfection, Transduction, Isolation, Over Expression

16) Product Images from "Novel genomes and genome constitutions identified by GISH and 5S rDNA and knotted1 genomic sequences in the genus Setaria"

Article Title: Novel genomes and genome constitutions identified by GISH and 5S rDNA and knotted1 genomic sequences in the genus Setaria

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-244

GISH patterns obtained on different Setaria species. ( a ) GISH was carried out using S. viridis -Q24 genomic DNA as probe hybridizing on the chromosome preparation of S. italica -Y1. ( b ) Metaphase of Qing 9 probed with S. viridis -Q24 total genomic DNA probe, two major spots were detected in the nucleolar organizing regions (arrows). ( c ) DAPI counterstained metaphase plate from S. adhaerans -W94. ( d ) The same metaphase plate was hybridized with Qing 9 genomic DNA (red). ( e ) The metaphase of Qing 9 was counterstained with DAPI. ( f ) The same metaphase hybridized with genomic DNA of S. adhaerans -W94 (red). ( g - h ) Genomic DNA of S. viridis -Q24 ( g ) and S. adhaerans -W94 ( h ) was applied to S. lachnea chromosomes respectively. ( i ) DAPI counterstained metaphase plate from S. lachnea . ( j ) The same metaphase hybridized with the total genomic DNA of S. grisebachii . ( k ) The metaphase of S. palmifolia was counterstained with DAPI. ( l ) The same metaphase plate was hybridized with the genomic DNA of S. plicata (red). ( m ) DAPI counterstained metaphase plate from S. parviflora -W79. ( n ) The same metaphase hybridized with S. glauca -W12 genomic DNA. ( o - r ) The metaphases of S. arenaria respectively hybridized with the genomic DNA of S. viridis -Q24 ( o ), S. adhaerans -W94 ( p ), S. grisebachii ( q ), and S. glauca -W12 ( r ). ( s ) Metaphase of S. palmifolia probed with S. viridis -Q24 total genomic DNA probe. ( t ) Metaphase plate from S. glauca -W12 hybridized with probe from S. grisebachii . Bar = 5 μm.
Figure Legend Snippet: GISH patterns obtained on different Setaria species. ( a ) GISH was carried out using S. viridis -Q24 genomic DNA as probe hybridizing on the chromosome preparation of S. italica -Y1. ( b ) Metaphase of Qing 9 probed with S. viridis -Q24 total genomic DNA probe, two major spots were detected in the nucleolar organizing regions (arrows). ( c ) DAPI counterstained metaphase plate from S. adhaerans -W94. ( d ) The same metaphase plate was hybridized with Qing 9 genomic DNA (red). ( e ) The metaphase of Qing 9 was counterstained with DAPI. ( f ) The same metaphase hybridized with genomic DNA of S. adhaerans -W94 (red). ( g - h ) Genomic DNA of S. viridis -Q24 ( g ) and S. adhaerans -W94 ( h ) was applied to S. lachnea chromosomes respectively. ( i ) DAPI counterstained metaphase plate from S. lachnea . ( j ) The same metaphase hybridized with the total genomic DNA of S. grisebachii . ( k ) The metaphase of S. palmifolia was counterstained with DAPI. ( l ) The same metaphase plate was hybridized with the genomic DNA of S. plicata (red). ( m ) DAPI counterstained metaphase plate from S. parviflora -W79. ( n ) The same metaphase hybridized with S. glauca -W12 genomic DNA. ( o - r ) The metaphases of S. arenaria respectively hybridized with the genomic DNA of S. viridis -Q24 ( o ), S. adhaerans -W94 ( p ), S. grisebachii ( q ), and S. glauca -W12 ( r ). ( s ) Metaphase of S. palmifolia probed with S. viridis -Q24 total genomic DNA probe. ( t ) Metaphase plate from S. glauca -W12 hybridized with probe from S. grisebachii . Bar = 5 μm.

Techniques Used:

17) Product Images from "A genetic switch controls the production of flagella and toxins in Clostridium difficile"

Article Title: A genetic switch controls the production of flagella and toxins in Clostridium difficile

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1006701

Assessing the purity of enriched populations. (A, B) Asymmetric PCR-digestion assay of genomic DNA to determine the orientation of the flagellar switch in individual colonies. Bacteria from individual colonies contain the switch predominantly in the published orientation (A) or the inverse orientation (B). Two representatives of each are shown. (C) Quantitative PCR results of the flagellar switch orientation in enriched phase variant populations (published, n = 9; inverse, n = 10). The ΔΔCt method was used to determine the relative DNA copies of the flagellar switch orientation in enriched populations relative to the rpoC gene.
Figure Legend Snippet: Assessing the purity of enriched populations. (A, B) Asymmetric PCR-digestion assay of genomic DNA to determine the orientation of the flagellar switch in individual colonies. Bacteria from individual colonies contain the switch predominantly in the published orientation (A) or the inverse orientation (B). Two representatives of each are shown. (C) Quantitative PCR results of the flagellar switch orientation in enriched phase variant populations (published, n = 9; inverse, n = 10). The ΔΔCt method was used to determine the relative DNA copies of the flagellar switch orientation in enriched populations relative to the rpoC gene.

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

Evidence for DNA inversion at the flagellar switch. (A) Nucleotide sequences corresponding to the 5’ UTR of the flgB operon from genome sequences available for PCR ribotype 027 strains were aligned using Clustal Omega. Shown are the regions corresponding to the putative flagellar switch and flanking imperfect inverted repeats. For strain BI1 “inverse”, the alignment was repeated after replacing the putative switch with its reverse complement. Identical nucleotides are indicated with blue shading. (B) Diagram of the PCR strategy used to detect the putative flagellar switch orientation. The primer names and sequences used for each strain are listed in the S2 Table . The predicted product sizes are based on R20291 sequence. (C) Orientation-specific PCR products for the flagellar switch from three C . difficile strains representing three ribotypes (R20291, 027, NCBI Accession No FN545816.1; ATCC43598, 017, NCBI sequence read archive SRX656590 [ 115 ]; 630Δ erm , 012, NCBI Accession No. EMBL:LN614756 [ 116 ]).
Figure Legend Snippet: Evidence for DNA inversion at the flagellar switch. (A) Nucleotide sequences corresponding to the 5’ UTR of the flgB operon from genome sequences available for PCR ribotype 027 strains were aligned using Clustal Omega. Shown are the regions corresponding to the putative flagellar switch and flanking imperfect inverted repeats. For strain BI1 “inverse”, the alignment was repeated after replacing the putative switch with its reverse complement. Identical nucleotides are indicated with blue shading. (B) Diagram of the PCR strategy used to detect the putative flagellar switch orientation. The primer names and sequences used for each strain are listed in the S2 Table . The predicted product sizes are based on R20291 sequence. (C) Orientation-specific PCR products for the flagellar switch from three C . difficile strains representing three ribotypes (R20291, 027, NCBI Accession No FN545816.1; ATCC43598, 017, NCBI sequence read archive SRX656590 [ 115 ]; 630Δ erm , 012, NCBI Accession No. EMBL:LN614756 [ 116 ]).

Techniques Used: Polymerase Chain Reaction, Sequencing

18) Product Images from "Reciprocal regulation of the basic helix-loop-helix/Per-Arnt-Sim partner proteins, Arnt and Arnt2, during neuronal differentiation"

Article Title: Reciprocal regulation of the basic helix-loop-helix/Per-Arnt-Sim partner proteins, Arnt and Arnt2, during neuronal differentiation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt206

Mapping the DNA methylation status of the Arnt2 promoter in P19 and Hepa1c1c7 cells. ( A ) CpG island prediction in the murine Arnt2 promoter using MethPrimer software. Criteria used for prediction were island size > 100 bp, GC percentage > 30% and observed/expected CpG ratio > 0.6. A single CpG island of 851 bp was identified, which spread across the transcriptional start site of the murine Arnt2 gene. ( B ) Bisulfite sequencing revealed the Arnt2 promoter was not methylated in either undifferentiated P19 cells (2d -RA) or 2 day and 4 day differentiated (i.e. 2d and 4d +RA treated) P19 cells, but was hypermethylated in hepatoma Hepa1c1c7 cells. The black circles indicate methylated CpG sites, and white circles indicate unmethylated CpG sites. PCR fragment analysed indicated by F and R boxes in (A).
Figure Legend Snippet: Mapping the DNA methylation status of the Arnt2 promoter in P19 and Hepa1c1c7 cells. ( A ) CpG island prediction in the murine Arnt2 promoter using MethPrimer software. Criteria used for prediction were island size > 100 bp, GC percentage > 30% and observed/expected CpG ratio > 0.6. A single CpG island of 851 bp was identified, which spread across the transcriptional start site of the murine Arnt2 gene. ( B ) Bisulfite sequencing revealed the Arnt2 promoter was not methylated in either undifferentiated P19 cells (2d -RA) or 2 day and 4 day differentiated (i.e. 2d and 4d +RA treated) P19 cells, but was hypermethylated in hepatoma Hepa1c1c7 cells. The black circles indicate methylated CpG sites, and white circles indicate unmethylated CpG sites. PCR fragment analysed indicated by F and R boxes in (A).

Techniques Used: DNA Methylation Assay, Software, Methylation Sequencing, Methylation, Polymerase Chain Reaction

19) Product Images from "The importance of DNA methylation of exons on alternative splicing"

Article Title: The importance of DNA methylation of exons on alternative splicing

Journal: RNA

doi: 10.1261/rna.064865.117

dCas9-TET1 targeting of DHODH exon 5 affects alternative splicing. ( A ) Schematic illustration of genomic region of DHODH from exon 4 to exon 6. Gray boxes and blue lines indicate exons and introns, respectively. Brown and black arrows represent sgRNA binding sites and RT-PCR primer binding sites, respectively. Red dashed lines indicate the region analyzed for DNA methylation. ( B ) Gel electrophoresis of RT-PCR products of the DHODH exon 5 region after overexpression of dCas9-TET1 with or without sgRNAs targeting exon 5 of DHODH gene in HCT116 cells. RNA was extracted, reverse transcribed, and primers were used to amplify the included (370 bp) or the skipped (172 bp) isoforms. NTC is non-template control. ( C ) HCT116 cells were either transfected with dCas9-TET1 only (control) or co-transfected with dCas9-TET1 and sgRNAs targeting different regions: DHODH exon 5 itself (+ex5 gRNA), DHODH intron 4 (+int4 gRNA), or DHODH ). Plotted are means ± SEM, (*) indicates P -value
Figure Legend Snippet: dCas9-TET1 targeting of DHODH exon 5 affects alternative splicing. ( A ) Schematic illustration of genomic region of DHODH from exon 4 to exon 6. Gray boxes and blue lines indicate exons and introns, respectively. Brown and black arrows represent sgRNA binding sites and RT-PCR primer binding sites, respectively. Red dashed lines indicate the region analyzed for DNA methylation. ( B ) Gel electrophoresis of RT-PCR products of the DHODH exon 5 region after overexpression of dCas9-TET1 with or without sgRNAs targeting exon 5 of DHODH gene in HCT116 cells. RNA was extracted, reverse transcribed, and primers were used to amplify the included (370 bp) or the skipped (172 bp) isoforms. NTC is non-template control. ( C ) HCT116 cells were either transfected with dCas9-TET1 only (control) or co-transfected with dCas9-TET1 and sgRNAs targeting different regions: DHODH exon 5 itself (+ex5 gRNA), DHODH intron 4 (+int4 gRNA), or DHODH ). Plotted are means ± SEM, (*) indicates P -value

Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, DNA Methylation Assay, Nucleic Acid Electrophoresis, Over Expression, Transfection

In vivo site-specific changes in DNA methylation induced by dCas9-DNMT3A-3L and dCas9-TET1. ( A ) Schematic illustration of EDI minigene. Gray and orange boxes indicate constitutive and alternative exons, respectively. Blue and black lines indicate introns and pFRT sequence, respectively. Black arrow represents the transcription start site (TSS). ( B ) Enlargement of EDI exon 5 region. Regions targeted by sgRNAs are indicated by brackets and letters above the gene schematic. Arrows below the gene schematic represent sgRNA binding sites. The arrow colors represent the different pools of sgRNAs used in panels C and D . Red dashed lines indicate the region analyzed for DNA methylation. ( C , D ) Methylation levels within exon 5 in ( C ) Met– cells transfected with dCas9-DNMT3A-3L or ( D ) Met+ cells transfected with dCas9-TET1 and a combination of three different sgRNAs (from the five or six sgRNAs designed to each region shown in panel B ). Different combinations of sgRNAs (marked #1, #2, or #3 below bars) were tested. “dCas9-DNMT3A-3L only” and “dCas9-TET1 only” are controls without sgRNAs and “untreated” indicates cells without transfection. (*) Indicates P -value
Figure Legend Snippet: In vivo site-specific changes in DNA methylation induced by dCas9-DNMT3A-3L and dCas9-TET1. ( A ) Schematic illustration of EDI minigene. Gray and orange boxes indicate constitutive and alternative exons, respectively. Blue and black lines indicate introns and pFRT sequence, respectively. Black arrow represents the transcription start site (TSS). ( B ) Enlargement of EDI exon 5 region. Regions targeted by sgRNAs are indicated by brackets and letters above the gene schematic. Arrows below the gene schematic represent sgRNA binding sites. The arrow colors represent the different pools of sgRNAs used in panels C and D . Red dashed lines indicate the region analyzed for DNA methylation. ( C , D ) Methylation levels within exon 5 in ( C ) Met– cells transfected with dCas9-DNMT3A-3L or ( D ) Met+ cells transfected with dCas9-TET1 and a combination of three different sgRNAs (from the five or six sgRNAs designed to each region shown in panel B ). Different combinations of sgRNAs (marked #1, #2, or #3 below bars) were tested. “dCas9-DNMT3A-3L only” and “dCas9-TET1 only” are controls without sgRNAs and “untreated” indicates cells without transfection. (*) Indicates P -value

Techniques Used: In Vivo, DNA Methylation Assay, Sequencing, Binding Assay, Methylation, Transfection

Schematic illustration of region-specific DNA methylation manipulations using the CRISPR system. Cells are co-transfected with a plasmid for expression of dCas9-DNMT3A-3L and the sgRNA expression plasmids. The dCas9 fusion protein is guided to a specific region of the genome by the sgRNAs where it brings DNMT3A-3L close to the DNA, resulting in site-specific methylation. As the transfection is transient, after several cell divisions, the fusion protein expression is lost but the DNA methylation pattern is maintained by the endogenous DNMT1 during genomic DNA replication. A similar method involving the dCas9-TET1 fusion enabled site-specific demethylation of DNA. The yellow spheres indicate methylated CpG.
Figure Legend Snippet: Schematic illustration of region-specific DNA methylation manipulations using the CRISPR system. Cells are co-transfected with a plasmid for expression of dCas9-DNMT3A-3L and the sgRNA expression plasmids. The dCas9 fusion protein is guided to a specific region of the genome by the sgRNAs where it brings DNMT3A-3L close to the DNA, resulting in site-specific methylation. As the transfection is transient, after several cell divisions, the fusion protein expression is lost but the DNA methylation pattern is maintained by the endogenous DNMT1 during genomic DNA replication. A similar method involving the dCas9-TET1 fusion enabled site-specific demethylation of DNA. The yellow spheres indicate methylated CpG.

Techniques Used: DNA Methylation Assay, CRISPR, Transfection, Plasmid Preparation, Expressing, Methylation

dCas9-TET1 induces DNA demethylation in a limited region. ( A ) Schematic illustration of EDI minigene exons 4 and 5. Exon 4 is alternatively spliced, whereas exon 5 is constitutively spliced. Blue and black lines indicate EDI introns and pFRT sequence, respectively. The six regions analyzed for methylation are indicated with letters. ( B ) The level of methylated CpGs (in percent) within the six regions denoted in panel A after transfection of dCas9-TET1 plasmid into Met+ cells with or without sgRNA expression (black and gray bars, respectively).
Figure Legend Snippet: dCas9-TET1 induces DNA demethylation in a limited region. ( A ) Schematic illustration of EDI minigene exons 4 and 5. Exon 4 is alternatively spliced, whereas exon 5 is constitutively spliced. Blue and black lines indicate EDI introns and pFRT sequence, respectively. The six regions analyzed for methylation are indicated with letters. ( B ) The level of methylated CpGs (in percent) within the six regions denoted in panel A after transfection of dCas9-TET1 plasmid into Met+ cells with or without sgRNA expression (black and gray bars, respectively).

Techniques Used: Sequencing, Methylation, Transfection, Plasmid Preparation, Expressing

20) Product Images from "Experimental induction of necrotic enteritis in chickens by anetB-positive Japanese isolate of Clostridiumperfringens"

Article Title: Experimental induction of necrotic enteritis in chickens by anetB-positive Japanese isolate of Clostridiumperfringens

Journal: The Journal of Veterinary Medical Science

doi: 10.1292/jvms.16-0500

PCR analysis of netB region of Clostridium perfringens isolate P-962. (A) result of a PCR assay with primers designed to amplify 963-, 1,829- and 2,012-bp PCR products corresponding to netB (2), the upstream (4) and downstream (5) fragments of netB gene. 100-bp ladder (1) and 1-kb (3) DNA markers are shown on the left of each gel. (B) Schematic diagram showing the location of each primer in P-962 DNA. The numbers with broken line indicate the length (kb) of PCR product amplified by the corresponding primers. The numbers with an arrow indicate kb.
Figure Legend Snippet: PCR analysis of netB region of Clostridium perfringens isolate P-962. (A) result of a PCR assay with primers designed to amplify 963-, 1,829- and 2,012-bp PCR products corresponding to netB (2), the upstream (4) and downstream (5) fragments of netB gene. 100-bp ladder (1) and 1-kb (3) DNA markers are shown on the left of each gel. (B) Schematic diagram showing the location of each primer in P-962 DNA. The numbers with broken line indicate the length (kb) of PCR product amplified by the corresponding primers. The numbers with an arrow indicate kb.

Techniques Used: Polymerase Chain Reaction, Amplification

21) Product Images from "The E3 ligase Mule protects the heart against oxidative stress and mitochondrial dysfunction through Myc-dependent inactivation of Pgc-1α and Pink1"

Article Title: The E3 ligase Mule protects the heart against oxidative stress and mitochondrial dysfunction through Myc-dependent inactivation of Pgc-1α and Pink1

Journal: Scientific Reports

doi: 10.1038/srep41490

Mule-deficiency induces cardiomyocyte apoptosis and structural remodeling of the ventricular wall. ( A ) Analysis of cardiac fibrosis by immunofluorescence microscopy employing wheat germ agglutinin (WGA) staining (green) of collagen deposition in the extracellular matrix, cardiomyocyte-specific anti-actinin (red), and Dapi (blue) to visualize nuclear DNA. ( B ) Quantification of extracellular matrix area indicative of LV fibrosis shown in ( B ). n = 4. ( C ) Acute genetic ablation of Mule triggers cardiomyocyte apoptosis which is abrogated by co- deletion of Mule and Myc in DKO mice. n = 4. ( D ) Analysis of apoptosis in LV cardiomyocytes (white arrows) by immunofluorescence microscopy and TUNEL assays. Hearts were harvested at 8 d post-Tam. Mice were 12 weeks old at the time of analysis. Red, cardiomyocyte-specific nuclear marker, anti-Mef2a. Green, TUNEL. Blue, DAPI stain of nuclear genomic DNA. TUNEL, terminal deoxynucleotidyl transferase- mediated dUTP nick-end-labeling. ( E ) BrdU, an indicator for DNA synthesis was injected intraperitoneally at 7 d post-Tam. Animals were sacrificed 18 hours later. Quantitative analysis of cardiomyocytes in S phase was performed by immunofluorescence microscopy of LV cardiac sections employing anti-BrdU (green) and anti-Mef2a (red) antibodies. White arrows denote BrdU-positive and Mef2a-negative non-cardiomyocytes. Blue, nuclei). BrdU, 5-Bromo-2′-deoxyuridine. ( F ) Genetic ablation of Mule fails to induce cell cycle entry and DNA synthesis in adult cardiomyocytes. n = 4. Data are means ± s.e.m.
Figure Legend Snippet: Mule-deficiency induces cardiomyocyte apoptosis and structural remodeling of the ventricular wall. ( A ) Analysis of cardiac fibrosis by immunofluorescence microscopy employing wheat germ agglutinin (WGA) staining (green) of collagen deposition in the extracellular matrix, cardiomyocyte-specific anti-actinin (red), and Dapi (blue) to visualize nuclear DNA. ( B ) Quantification of extracellular matrix area indicative of LV fibrosis shown in ( B ). n = 4. ( C ) Acute genetic ablation of Mule triggers cardiomyocyte apoptosis which is abrogated by co- deletion of Mule and Myc in DKO mice. n = 4. ( D ) Analysis of apoptosis in LV cardiomyocytes (white arrows) by immunofluorescence microscopy and TUNEL assays. Hearts were harvested at 8 d post-Tam. Mice were 12 weeks old at the time of analysis. Red, cardiomyocyte-specific nuclear marker, anti-Mef2a. Green, TUNEL. Blue, DAPI stain of nuclear genomic DNA. TUNEL, terminal deoxynucleotidyl transferase- mediated dUTP nick-end-labeling. ( E ) BrdU, an indicator for DNA synthesis was injected intraperitoneally at 7 d post-Tam. Animals were sacrificed 18 hours later. Quantitative analysis of cardiomyocytes in S phase was performed by immunofluorescence microscopy of LV cardiac sections employing anti-BrdU (green) and anti-Mef2a (red) antibodies. White arrows denote BrdU-positive and Mef2a-negative non-cardiomyocytes. Blue, nuclei). BrdU, 5-Bromo-2′-deoxyuridine. ( F ) Genetic ablation of Mule fails to induce cell cycle entry and DNA synthesis in adult cardiomyocytes. n = 4. Data are means ± s.e.m.

Techniques Used: Immunofluorescence, Microscopy, Whole Genome Amplification, Staining, Mouse Assay, TUNEL Assay, Marker, End Labeling, DNA Synthesis, Injection

22) Product Images from "Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors"

Article Title: Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors

Journal: BMC Genomics

doi: 10.1186/s12864-015-1321-y

Detection of the circular features of the three plasmids by PFGE. (A-C) Plasmid detection by PFGE separation and Southern blot hybridization. A , hybridization with lcp3-specific probes after PFGE separation; B , hybridization with lcp2-specific probes after PFGE separation; C , hybridization with lcp1-specific probes after PFGE separation. The left side of each picture represents PFGE separation of genomic DNA while the right side represents Southern blot hybridization with plasmid-specific probes; M, bacteriophage λ DNA multimer marker (monomer =48.5 kb). ND, undigested DNA; Asc I, Not I, BssH II, Sma I, and Xho I represent L. interrogans strain 56609 digested with these enzymes. L. interrogans serovar Lai strain 56601 genomic DNA was also blotted onto a nylon membrane as a negative control. The primers for the synthetized probes are listed in Additional file 1 : Table S3.
Figure Legend Snippet: Detection of the circular features of the three plasmids by PFGE. (A-C) Plasmid detection by PFGE separation and Southern blot hybridization. A , hybridization with lcp3-specific probes after PFGE separation; B , hybridization with lcp2-specific probes after PFGE separation; C , hybridization with lcp1-specific probes after PFGE separation. The left side of each picture represents PFGE separation of genomic DNA while the right side represents Southern blot hybridization with plasmid-specific probes; M, bacteriophage λ DNA multimer marker (monomer =48.5 kb). ND, undigested DNA; Asc I, Not I, BssH II, Sma I, and Xho I represent L. interrogans strain 56609 digested with these enzymes. L. interrogans serovar Lai strain 56601 genomic DNA was also blotted onto a nylon membrane as a negative control. The primers for the synthetized probes are listed in Additional file 1 : Table S3.

Techniques Used: Plasmid Preparation, Southern Blot, Hybridization, Marker, Negative Control

23) Product Images from "Persistent inhibition of telomerase reprograms adult T-cell leukemia to p53-dependent senescence"

Article Title: Persistent inhibition of telomerase reprograms adult T-cell leukemia to p53-dependent senescence

Journal: Blood

doi: 10.1182/blood-2006-01-0067

Analysis of CD25 + HTLV-I–infected cells. (A) FACS analysis of the presence of CD25 marker expression from CD25 + and CD25– sorted cells. (B) Telomerase activity detected by TRAP assay in CD25 + and CD25– fractions isolated from an HTLV-I donor and ATL PBMCs. (C) Integrated HTLV-I proviral DNA was detected by PCR using primers in the tax coding region. GAPDH was used as amplification control to ensure proper quality and quantity of extracted DNAs. (D) Staining control of PBMCs in presence or absence of FITC-telomere probe. (E) In situ hybridization of FITC-conjugated telomere probe (top) and DAPI (4′6-diamidino-2-phenylindole 2HCl) (bottom).
Figure Legend Snippet: Analysis of CD25 + HTLV-I–infected cells. (A) FACS analysis of the presence of CD25 marker expression from CD25 + and CD25– sorted cells. (B) Telomerase activity detected by TRAP assay in CD25 + and CD25– fractions isolated from an HTLV-I donor and ATL PBMCs. (C) Integrated HTLV-I proviral DNA was detected by PCR using primers in the tax coding region. GAPDH was used as amplification control to ensure proper quality and quantity of extracted DNAs. (D) Staining control of PBMCs in presence or absence of FITC-telomere probe. (E) In situ hybridization of FITC-conjugated telomere probe (top) and DAPI (4′6-diamidino-2-phenylindole 2HCl) (bottom).

Techniques Used: Infection, FACS, Marker, Expressing, Activity Assay, TRAP Assay, Isolation, Polymerase Chain Reaction, Amplification, Staining, In Situ Hybridization

24) Product Images from "Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells"

Article Title: Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-11418-3

Isolation of miR-125a promoter. ( A ) Two major transcripts of SPACA6 gene and map of its genomic locus; exons and microRNAs are indicated by black boxes and loops, respectively; the first base of pre-miR-99b was assigned as nucleotide 1 ( B ) Five genomic DNA segments (grey bars in the left side of the panel) spanning nucleotides -36 to -3875 were cloned in the luciferase reporter plasmid pGL3-basic and assayed for transcription promoter activity in HepG2 cells. The reporter constructs are named according to the size of the cloned genomic fragment and their activity is reported in the adjacent plot; 869mut construct carries two point mutations (marked by white dots) eliminating putative translation start sites. Assays were performed at least in triplicate and expressed as mean ± SD.
Figure Legend Snippet: Isolation of miR-125a promoter. ( A ) Two major transcripts of SPACA6 gene and map of its genomic locus; exons and microRNAs are indicated by black boxes and loops, respectively; the first base of pre-miR-99b was assigned as nucleotide 1 ( B ) Five genomic DNA segments (grey bars in the left side of the panel) spanning nucleotides -36 to -3875 were cloned in the luciferase reporter plasmid pGL3-basic and assayed for transcription promoter activity in HepG2 cells. The reporter constructs are named according to the size of the cloned genomic fragment and their activity is reported in the adjacent plot; 869mut construct carries two point mutations (marked by white dots) eliminating putative translation start sites. Assays were performed at least in triplicate and expressed as mean ± SD.

Techniques Used: Isolation, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Construct

25) Product Images from "A single digital droplet PCR assay to detect multiple KIT exon 11 mutations in tumor and plasma from patients with gastrointestinal stromal tumors"

Article Title: A single digital droplet PCR assay to detect multiple KIT exon 11 mutations in tumor and plasma from patients with gastrointestinal stromal tumors

Journal: Oncotarget

doi: 10.18632/oncotarget.24493

Detection of KIT exon 11 mutations using the ddPCR drop-off assay in ctDNA in patients with metastasized GIST at baseline (before start TKI-treatment) and 2–6 weeks after start of treatment Mutation frequency is expressed as fractional abundance in % (see Supplementary Table 1 ). Twelve patients with metastasized GIST with both a baseline plasma sample as well as at least one sample collected 2–6 weeks after staring TKI treatment were selected. Both pre-treatment FFPE DNA (Table 1 ) and baseline plasma samples (Table 2 ) were tested with the same ddPCR. Patient 39 and 41 (Table 3 ) were not included since no follow-up plasma samples were available.
Figure Legend Snippet: Detection of KIT exon 11 mutations using the ddPCR drop-off assay in ctDNA in patients with metastasized GIST at baseline (before start TKI-treatment) and 2–6 weeks after start of treatment Mutation frequency is expressed as fractional abundance in % (see Supplementary Table 1 ). Twelve patients with metastasized GIST with both a baseline plasma sample as well as at least one sample collected 2–6 weeks after staring TKI treatment were selected. Both pre-treatment FFPE DNA (Table 1 ) and baseline plasma samples (Table 2 ) were tested with the same ddPCR. Patient 39 and 41 (Table 3 ) were not included since no follow-up plasma samples were available.

Techniques Used: Mutagenesis, Formalin-fixed Paraffin-Embedded

26) Product Images from "Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference"

Article Title: Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkz748

Identities of cells that require RRF-1 for silencing by neuronal dsRNA vary from animal to animal. ( A ) GFP expression from the sur-5::gfp chimeric gene enables simultaneous visualization of most somatic nuclei in C. elegans . A depth coded (one color for ∼53 frames) projection of 5 Z-stacks that were stitched together from a single L4-staged animal is shown (also see Materials and Methods). Scale bar = 100 μm. ( B-D ) Expression of gfp -dsRNA in neurons causes silencing throughout the animal that is entirely dependent on SID-1 and partially dependent on RRF-1. Representative images of L4-staged sur-5::gfp animals that express Prgef-1::gfp -dsRNA (B) and additionally lack sid-1 (C) or rrf-1 (D) are shown. Maximum intensity projections of sections were stitched together to generate whole-worm images. Presence of gfp- dsRNA causes worms to twist because of the rol-6 co-injection marker. Cells that require RRF-1 for silencing (e.g. the excretory canal cell indicated by red arrows and some intestinal cells in red dashed lines) and cells that can silence in the absence of RRF-1 (e.g. some intestinal cells shown in blue dashed lines) are highlighted in Prgef-1::gfp- dsRNA ; rrf-1(-) and in Prgef-1::gfp -dsRNA; sid-1(-) animals. Scale bar = 100μm. ( E, F ) Silencing in rrf-1(- ) animals by neuronal dsRNA is associated with a decrease in sur-5::gfp mRNA levels. Single molecule FISH was used to detect sur-5::gfp mRNA in L4-staged wild-type animals (E, left ) or in sur-5::gfp animals that express Prgef-1::gfp- dsRNA (E, middle ) and that in addition lack sid-1 (E, right ) or rrf-1 (F). RNA from Prgef-1::gfp- dsRNA is prominently detected by gfp probes in neuronal nuclei (red arrows and bracket). A representative pair of intestinal nuclei is shown for each animal as an overlay of DNA (DAPI in blue), mRNA ( gfp in green) and protein (GFP in magenta). Cytoplasmic mRNA foci were counted (see Materials and Methods) between two nuclei in wild-type or in sid-1(-) backgrounds (E), and between two nuclei where GFP is silenced (off, blue) and where GFP is expressed (on, red) in rrf-1(-) animals. Errors indicate 95% confidence intervals, n = 3 in E and n = 4 in F. Top scale bar = 10 μm and bottom scale bar = 5 μm. ( G ) No intestinal cell requires RRF-1 for silencing in all animals. The E blastomere divides to generate 20 intestinal cells (EaLAAD to EpRPPP). Of the 20 cells, 10 undergo nuclear division without cell division (two grey circles per cell), 4 sometimes undergo similar nuclear division (one grey circle and one open circle per cell), and 6 do not undergo any division (one grey circle per cell). In each of 10 sur-5::gfp; rrf-1(-); Prgef-1::gfp -dsRNA L4-staged animals, GFP-positive nuclei (use only RRF-1, gray) and GFP-negative nuclei (use EGO-1 or RRF-1, white) were scored. Every binucleate cell had both nuclei with the same requirement. White boxes with a slash indicate absence of second nucleus because of lack of nuclear division ( 52 ). See Supplementary Figure S7 for images of additional animals.
Figure Legend Snippet: Identities of cells that require RRF-1 for silencing by neuronal dsRNA vary from animal to animal. ( A ) GFP expression from the sur-5::gfp chimeric gene enables simultaneous visualization of most somatic nuclei in C. elegans . A depth coded (one color for ∼53 frames) projection of 5 Z-stacks that were stitched together from a single L4-staged animal is shown (also see Materials and Methods). Scale bar = 100 μm. ( B-D ) Expression of gfp -dsRNA in neurons causes silencing throughout the animal that is entirely dependent on SID-1 and partially dependent on RRF-1. Representative images of L4-staged sur-5::gfp animals that express Prgef-1::gfp -dsRNA (B) and additionally lack sid-1 (C) or rrf-1 (D) are shown. Maximum intensity projections of sections were stitched together to generate whole-worm images. Presence of gfp- dsRNA causes worms to twist because of the rol-6 co-injection marker. Cells that require RRF-1 for silencing (e.g. the excretory canal cell indicated by red arrows and some intestinal cells in red dashed lines) and cells that can silence in the absence of RRF-1 (e.g. some intestinal cells shown in blue dashed lines) are highlighted in Prgef-1::gfp- dsRNA ; rrf-1(-) and in Prgef-1::gfp -dsRNA; sid-1(-) animals. Scale bar = 100μm. ( E, F ) Silencing in rrf-1(- ) animals by neuronal dsRNA is associated with a decrease in sur-5::gfp mRNA levels. Single molecule FISH was used to detect sur-5::gfp mRNA in L4-staged wild-type animals (E, left ) or in sur-5::gfp animals that express Prgef-1::gfp- dsRNA (E, middle ) and that in addition lack sid-1 (E, right ) or rrf-1 (F). RNA from Prgef-1::gfp- dsRNA is prominently detected by gfp probes in neuronal nuclei (red arrows and bracket). A representative pair of intestinal nuclei is shown for each animal as an overlay of DNA (DAPI in blue), mRNA ( gfp in green) and protein (GFP in magenta). Cytoplasmic mRNA foci were counted (see Materials and Methods) between two nuclei in wild-type or in sid-1(-) backgrounds (E), and between two nuclei where GFP is silenced (off, blue) and where GFP is expressed (on, red) in rrf-1(-) animals. Errors indicate 95% confidence intervals, n = 3 in E and n = 4 in F. Top scale bar = 10 μm and bottom scale bar = 5 μm. ( G ) No intestinal cell requires RRF-1 for silencing in all animals. The E blastomere divides to generate 20 intestinal cells (EaLAAD to EpRPPP). Of the 20 cells, 10 undergo nuclear division without cell division (two grey circles per cell), 4 sometimes undergo similar nuclear division (one grey circle and one open circle per cell), and 6 do not undergo any division (one grey circle per cell). In each of 10 sur-5::gfp; rrf-1(-); Prgef-1::gfp -dsRNA L4-staged animals, GFP-positive nuclei (use only RRF-1, gray) and GFP-negative nuclei (use EGO-1 or RRF-1, white) were scored. Every binucleate cell had both nuclei with the same requirement. White boxes with a slash indicate absence of second nucleus because of lack of nuclear division ( 52 ). See Supplementary Figure S7 for images of additional animals.

Techniques Used: Expressing, Injection, Marker, Fluorescence In Situ Hybridization

27) Product Images from "Gene promoter and exon DNA methylation changes in colon cancer development – mRNA expression and tumor mutation alterations"

Article Title: Gene promoter and exon DNA methylation changes in colon cancer development – mRNA expression and tumor mutation alterations

Journal: BMC Cancer

doi: 10.1186/s12885-018-4609-x

DNA methylation alterations in mutation hot-spot regions of genes frequently mutated in CRC and adenoma. Methylation percentage values are shown in 100 base pair analysis regions located in mutation hot-spot areas of genes ( TP53, APC, KRAS, BRAF and FBXW7 ) frequently mutated in CRC and adenoma tissue. The frequencies of mutations in CRC and adenoma samples detected in our previous multiplex PCR-based CRC mutation hot-spot sequencing study [ 27 ] are also represented. * p
Figure Legend Snippet: DNA methylation alterations in mutation hot-spot regions of genes frequently mutated in CRC and adenoma. Methylation percentage values are shown in 100 base pair analysis regions located in mutation hot-spot areas of genes ( TP53, APC, KRAS, BRAF and FBXW7 ) frequently mutated in CRC and adenoma tissue. The frequencies of mutations in CRC and adenoma samples detected in our previous multiplex PCR-based CRC mutation hot-spot sequencing study [ 27 ] are also represented. * p

Techniques Used: DNA Methylation Assay, Mutagenesis, Methylation, Multiplex Assay, Polymerase Chain Reaction, Sequencing

28) Product Images from "Diversifying Sunflower Germplasm by Integration and Mapping of a Novel Male Fertility Restoration Gene"

Article Title: Diversifying Sunflower Germplasm by Integration and Mapping of a Novel Male Fertility Restoration Gene

Journal: Genetics

doi: 10.1534/genetics.112.146092

Genomic in situ hybridization (GISH) analyses of the alien H. angustifolius chromosome or segments in different progenies. The genomic DNA of H. angustifolius was labeled with digoxigenin–11-dUTP and detected by anti-dig-rhodamine (red), the chromosomes
Figure Legend Snippet: Genomic in situ hybridization (GISH) analyses of the alien H. angustifolius chromosome or segments in different progenies. The genomic DNA of H. angustifolius was labeled with digoxigenin–11-dUTP and detected by anti-dig-rhodamine (red), the chromosomes

Techniques Used: In Situ Hybridization, Labeling

29) Product Images from "AID induces intraclonal diversity and genomic damage in CD86+ chronic lymphocytic leukemia cells"

Article Title: AID induces intraclonal diversity and genomic damage in CD86+ chronic lymphocytic leukemia cells

Journal: European Journal of Immunology

doi: 10.1002/eji.201344421

Sequence analysis of IgV and Sμ regions of two IgV-UM and two IgV-Mut CLL samples. (A) From four purified CLL samples, DNA was extracted and subjected to nested PCR to amplify and deep sequence the VDJ and Sμ regions from the rearranged allele. A schematic representation of the rearranged IgH locus is indicated, showing the VDJ gene, the Iμ exon, the Sμ region, and the first constant exon of the IgM heavy chain (CH1μ). Primer-binding sites are indicated (gray: first round PCR and black: second round PCR). The graphs show the set of subclonal VDJ and Sμ sequences (seq#) appearing within the individual samples with a frequency above 0.1%. The particular VDJ usage is indicated below each sample name. Dots within graphs display the position of bases that do not match with the dominant sequence. Base substitutions corresponding to germ line sequences in Sμ are indicated with x. IgV mutations of the dominant clone (D) are indicated as vertical bars along the x -axis for the two IgV-Mut samples. (All sequences are listed in Supporting Information Table 3 .) (B) The frequency of subclonal sequences was determined by dividing the number of sublonal sequences gained by next generation sequencing (NGS) by the number of total sequences. The number above each pie chart gives the percentage of all subclonal variations of the respective sequence (only sequences with a frequency > 0.1% were counted). The mean percentage of base variants/sequence for all IgV versus Sμ regions and the resulting p -value are indicated on the bottom of the graph. Data are compiled from one NGS experiment on four samples (#1–4). (C) Replacement mutations at VDJ genes of all subclonal variations shown in (A) were determined according to the genetic letter code. Nonsense mutations are marked with an asterisk. (D) The mutation spectrum for all VDJ and Sμ mutations depicted in (A) is shown. Nucleotides are listed on the axes and the numbers in each box represent the number of the respective mutation.
Figure Legend Snippet: Sequence analysis of IgV and Sμ regions of two IgV-UM and two IgV-Mut CLL samples. (A) From four purified CLL samples, DNA was extracted and subjected to nested PCR to amplify and deep sequence the VDJ and Sμ regions from the rearranged allele. A schematic representation of the rearranged IgH locus is indicated, showing the VDJ gene, the Iμ exon, the Sμ region, and the first constant exon of the IgM heavy chain (CH1μ). Primer-binding sites are indicated (gray: first round PCR and black: second round PCR). The graphs show the set of subclonal VDJ and Sμ sequences (seq#) appearing within the individual samples with a frequency above 0.1%. The particular VDJ usage is indicated below each sample name. Dots within graphs display the position of bases that do not match with the dominant sequence. Base substitutions corresponding to germ line sequences in Sμ are indicated with x. IgV mutations of the dominant clone (D) are indicated as vertical bars along the x -axis for the two IgV-Mut samples. (All sequences are listed in Supporting Information Table 3 .) (B) The frequency of subclonal sequences was determined by dividing the number of sublonal sequences gained by next generation sequencing (NGS) by the number of total sequences. The number above each pie chart gives the percentage of all subclonal variations of the respective sequence (only sequences with a frequency > 0.1% were counted). The mean percentage of base variants/sequence for all IgV versus Sμ regions and the resulting p -value are indicated on the bottom of the graph. Data are compiled from one NGS experiment on four samples (#1–4). (C) Replacement mutations at VDJ genes of all subclonal variations shown in (A) were determined according to the genetic letter code. Nonsense mutations are marked with an asterisk. (D) The mutation spectrum for all VDJ and Sμ mutations depicted in (A) is shown. Nucleotides are listed on the axes and the numbers in each box represent the number of the respective mutation.

Techniques Used: Sequencing, Purification, Nested PCR, Binding Assay, Polymerase Chain Reaction, Next-Generation Sequencing, Mutagenesis

IgV and Sμ region mutations in unsorted versus CD86 + -sorted CLL cells. The diverse set of VDJ sequences (seq#) for (A) IgV-Mut #3 and (B) IgV-Mut #4, appearing with a frequency > 0.1%, is shown for unsorted and CD86 + -sorted CLL cells as described in Figure 1 . (For IgV-Mut #4, only sequences from CD86 + -sorted samples are shown as no subclonal variations were detected in unsorted samples.) The relative frequencies of the subclones are indicated as horizontal bars on the right of each graph. Data are compiled from one NGS experiment on pooled tagged amplicons derived from DNA of > 50 000 sorted cells. Sequences of subclonal VDJ mutations from CD86 + -sorted samples of (C) IgV-Mut #3 and (D) IgV-Mut #4 are shown in alignment with germ line V sequences (GL) and the respective dominant clone (D). Amino acid changes are shown underneath the alignment and are indicated in gray. Silent mutations are underlined. The resulting mutation spectrum is shown in (E). Nucleotides are listed on the axes and the numbers in each box represent the number of the respective mutation. (F) Genealogical relation of VDJ mutations from CD86 + -sorted IgV-Mut #4 is given. The frequency (frq) of the individual subclones is indicated. The branch support values are given within the diagram and the length of each branch is proportional to the number of varying bases (evolutionary distance).
Figure Legend Snippet: IgV and Sμ region mutations in unsorted versus CD86 + -sorted CLL cells. The diverse set of VDJ sequences (seq#) for (A) IgV-Mut #3 and (B) IgV-Mut #4, appearing with a frequency > 0.1%, is shown for unsorted and CD86 + -sorted CLL cells as described in Figure 1 . (For IgV-Mut #4, only sequences from CD86 + -sorted samples are shown as no subclonal variations were detected in unsorted samples.) The relative frequencies of the subclones are indicated as horizontal bars on the right of each graph. Data are compiled from one NGS experiment on pooled tagged amplicons derived from DNA of > 50 000 sorted cells. Sequences of subclonal VDJ mutations from CD86 + -sorted samples of (C) IgV-Mut #3 and (D) IgV-Mut #4 are shown in alignment with germ line V sequences (GL) and the respective dominant clone (D). Amino acid changes are shown underneath the alignment and are indicated in gray. Silent mutations are underlined. The resulting mutation spectrum is shown in (E). Nucleotides are listed on the axes and the numbers in each box represent the number of the respective mutation. (F) Genealogical relation of VDJ mutations from CD86 + -sorted IgV-Mut #4 is given. The frequency (frq) of the individual subclones is indicated. The branch support values are given within the diagram and the length of each branch is proportional to the number of varying bases (evolutionary distance).

Techniques Used: Next-Generation Sequencing, Derivative Assay, Mutagenesis

CD86 + cells represent the proliferative CLL fraction. (A) Representative flow cytometry profile of a CLL sample showing CXCR4 and CD86 expression on CD5 + CD19 + pregated CLL cells. Boxes and numbers in dot plots indicate gating for CD86 positive versus negative cells. Plots are representative of 50 independent experiments. (B) MFI values of CXCR4 + cells are quantified for CD86 – and CD86 + CLL cells (pregated for CD5 + CD19 + expression; each individual patient sample is represented by a single circle symbol (CD86 − fraction) and quadrant symbol (CD86 + fraction), n = 50; mean values shown as horizontal lines; p -value determined by paired t -test). (C) Representative flow cytometry profile for CD86/Ki67 coexpression on CD5 + CD19 + pregated CLL cells. Boxes and numbers in dot plots indicate percentages of cells within the respective quadrants. Plots are representative of 38 independent experiments. (D) The ratio of Ki67 + /Ki67 − CLL cells within CD86 − and CD86 + subsets was determined by flow cytometry based on the quadrant percentages shown in (C). Each individual patient sample is represented by a single circle symbol (CD86 − fraction) and quadrant symbol (CD86 + fraction), n = 38; mean values shown as horizontal lines; p -value determined by Mann–Whitney test. (E) Representative cell cycle profiles of CD86 − and CD86 + CLL fractions (pregated on CD5 + CD19 + cells) of patient ID 565, determined by DAPI DNA staining. Percentages are shown within the FACS graphs. Histograms are representative of five independent experiments. (F) Cell cycle profiles were analyzed by plotting the percentages of CLL cells within the respective cell cycle phase as determined in (E) for CD86 − (black bars) and CD86 + (white bars) CLL cells; (div = dividing cells in M/S/G2; n = 5; bars show means ± SD; p -values determined by Mann–Whitney test). (G) Flow cytometry was used to examine the expression pattern of CD5 and CXCR4 on CD86 + CD5 + CD19 + CLL cells (blue) within the total population of CD5 + CD19 + CLL cells (red). Dot plots are representative of 50 independent experiments.
Figure Legend Snippet: CD86 + cells represent the proliferative CLL fraction. (A) Representative flow cytometry profile of a CLL sample showing CXCR4 and CD86 expression on CD5 + CD19 + pregated CLL cells. Boxes and numbers in dot plots indicate gating for CD86 positive versus negative cells. Plots are representative of 50 independent experiments. (B) MFI values of CXCR4 + cells are quantified for CD86 – and CD86 + CLL cells (pregated for CD5 + CD19 + expression; each individual patient sample is represented by a single circle symbol (CD86 − fraction) and quadrant symbol (CD86 + fraction), n = 50; mean values shown as horizontal lines; p -value determined by paired t -test). (C) Representative flow cytometry profile for CD86/Ki67 coexpression on CD5 + CD19 + pregated CLL cells. Boxes and numbers in dot plots indicate percentages of cells within the respective quadrants. Plots are representative of 38 independent experiments. (D) The ratio of Ki67 + /Ki67 − CLL cells within CD86 − and CD86 + subsets was determined by flow cytometry based on the quadrant percentages shown in (C). Each individual patient sample is represented by a single circle symbol (CD86 − fraction) and quadrant symbol (CD86 + fraction), n = 38; mean values shown as horizontal lines; p -value determined by Mann–Whitney test. (E) Representative cell cycle profiles of CD86 − and CD86 + CLL fractions (pregated on CD5 + CD19 + cells) of patient ID 565, determined by DAPI DNA staining. Percentages are shown within the FACS graphs. Histograms are representative of five independent experiments. (F) Cell cycle profiles were analyzed by plotting the percentages of CLL cells within the respective cell cycle phase as determined in (E) for CD86 − (black bars) and CD86 + (white bars) CLL cells; (div = dividing cells in M/S/G2; n = 5; bars show means ± SD; p -values determined by Mann–Whitney test). (G) Flow cytometry was used to examine the expression pattern of CD5 and CXCR4 on CD86 + CD5 + CD19 + CLL cells (blue) within the total population of CD5 + CD19 + CLL cells (red). Dot plots are representative of 50 independent experiments.

Techniques Used: Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY, Staining, FACS

30) Product Images from "Regulation of cellular growth by the Drosophila target of rapamycin dTOR"

Article Title: Regulation of cellular growth by the Drosophila target of rapamycin dTOR

Journal: Genes & Development

doi:

Mutations in dTOR inhibit larval growth. ( A ) The structure of the 8.0-kb dTOR transcript is shown. Coding sequence is indicated by filled boxes; 5′ and 3′ untranslated regions are represented by open boxes. Breaks in the boxes indicate introns. Sites of the two P-element insertions in dTOR are indicated, and the range of the dTOR ΔP deletion is shown. The stippled bar at the bottom of the figure indicates the DNA fragment used for genomic rescue. Vha68-1 encodes a mitochondrial ATPase transcribed in the opposite direction as dTOR . ( B ) Size comparison of wild-type and dTOR homozygous mutant larvae at 126 h of development. Wild-type larvae pupate within 12 h of this time point, whereas dTOR mutants remain arrested in the larval stage with little or no further growth. ( C ) Heterozygosity for dTOR sensitizes larvae to rapamycin. On normal food, wild-type (black circles) and dTOR /+ larvae (black diamonds) grow at indistinguishable rates. Addition of 1 μM rapamycin delays development by ∼3 d in wild-type (open circles) and ∼6 d in dTOR /+ larvae (open diamonds).
Figure Legend Snippet: Mutations in dTOR inhibit larval growth. ( A ) The structure of the 8.0-kb dTOR transcript is shown. Coding sequence is indicated by filled boxes; 5′ and 3′ untranslated regions are represented by open boxes. Breaks in the boxes indicate introns. Sites of the two P-element insertions in dTOR are indicated, and the range of the dTOR ΔP deletion is shown. The stippled bar at the bottom of the figure indicates the DNA fragment used for genomic rescue. Vha68-1 encodes a mitochondrial ATPase transcribed in the opposite direction as dTOR . ( B ) Size comparison of wild-type and dTOR homozygous mutant larvae at 126 h of development. Wild-type larvae pupate within 12 h of this time point, whereas dTOR mutants remain arrested in the larval stage with little or no further growth. ( C ) Heterozygosity for dTOR sensitizes larvae to rapamycin. On normal food, wild-type (black circles) and dTOR /+ larvae (black diamonds) grow at indistinguishable rates. Addition of 1 μM rapamycin delays development by ∼3 d in wild-type (open circles) and ∼6 d in dTOR /+ larvae (open diamonds).

Techniques Used: Sequencing, Mutagenesis

31) Product Images from "Immortalization of human AE pre-leukemia cells by hTERT allows leukemic transformation"

Article Title: Immortalization of human AE pre-leukemia cells by hTERT allows leukemic transformation

Journal: Oncotarget

doi: 10.18632/oncotarget.11093

AE pre-leukemia cells are immortalized by hTERT A. hTERT mRNA analyzed by qPCR in CD34+HSPC transduced with AE or control empty vector (MIG). Error bar represents SD, n = 4. B. Telomerase activity of control AE, AE-hTERT and Kasumi-1 cells. Cell extracts heated (HT) to inactivate telomerase were used as negative control. C. Weekly cell count of AE-hTERT and control AE cells. D. Telomere length of AE-hTERT and control cells from culture of different time points measured by southern blot with a telomeric probe. E. Telomere FISH analysis by telomere specific DNA probe on week 26 AE-hTERT and AE-pBabe cells. Representative cells at metaphase are shown, telomere-free chromosome ends are indicated by arrow. 30 metaphases for each sample were scored, and average number of telomere-free chromosome ends were indicated ( p
Figure Legend Snippet: AE pre-leukemia cells are immortalized by hTERT A. hTERT mRNA analyzed by qPCR in CD34+HSPC transduced with AE or control empty vector (MIG). Error bar represents SD, n = 4. B. Telomerase activity of control AE, AE-hTERT and Kasumi-1 cells. Cell extracts heated (HT) to inactivate telomerase were used as negative control. C. Weekly cell count of AE-hTERT and control AE cells. D. Telomere length of AE-hTERT and control cells from culture of different time points measured by southern blot with a telomeric probe. E. Telomere FISH analysis by telomere specific DNA probe on week 26 AE-hTERT and AE-pBabe cells. Representative cells at metaphase are shown, telomere-free chromosome ends are indicated by arrow. 30 metaphases for each sample were scored, and average number of telomere-free chromosome ends were indicated ( p

Techniques Used: Real-time Polymerase Chain Reaction, Transduction, Plasmid Preparation, Activity Assay, Negative Control, Cell Counting, Southern Blot, Fluorescence In Situ Hybridization

32) Product Images from "Whole Genome Incorporation and Epigenetic Stability in a Newly Synthetic Allopolyploid of Gynogenetic Gibel Carp"

Article Title: Whole Genome Incorporation and Epigenetic Stability in a Newly Synthetic Allopolyploid of Gynogenetic Gibel Carp

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evy165

—Sequence comparison and localizations of 45s rDNAs. ( A ) Sequence comparison of 45s rDNAs in allopolyploid (allo) (allo-45S1: MH290789; allo-45S2: MH290791), gibel carp clone D (hexa) (MH290792), and common carp ( Cc ) (MH290790). The primer sequences had been excluded. ( B ) Localizations of 45s rDNA in Cc ( B ), hex ( C ), and allo ( D ) by FISH with 45S rDNA probe. ( E ) Dual localizations of 45S rDNA labeled with digoxigenin and chromosomes from Cc labeled with biotin in a metaphase chromosome spread of allopolyploid. All metaphase chromosomes were counterstained with DAPI and the red fluorescene signals showed 50 chromosomes from common carp in allopolyploid. The white arrows pointing to the green fluorescence indicate the 45S rDNA loci, and the red arrow indicates colabeled loci by 45S rDNA probe and common carp genomic DNA probe.
Figure Legend Snippet: —Sequence comparison and localizations of 45s rDNAs. ( A ) Sequence comparison of 45s rDNAs in allopolyploid (allo) (allo-45S1: MH290789; allo-45S2: MH290791), gibel carp clone D (hexa) (MH290792), and common carp ( Cc ) (MH290790). The primer sequences had been excluded. ( B ) Localizations of 45s rDNA in Cc ( B ), hex ( C ), and allo ( D ) by FISH with 45S rDNA probe. ( E ) Dual localizations of 45S rDNA labeled with digoxigenin and chromosomes from Cc labeled with biotin in a metaphase chromosome spread of allopolyploid. All metaphase chromosomes were counterstained with DAPI and the red fluorescene signals showed 50 chromosomes from common carp in allopolyploid. The white arrows pointing to the green fluorescence indicate the 45S rDNA loci, and the red arrow indicates colabeled loci by 45S rDNA probe and common carp genomic DNA probe.

Techniques Used: Sequencing, Fluorescence In Situ Hybridization, Labeling, Fluorescence

33) Product Images from "Arachis batizocoi: a study of its relationship to cultivated peanut (A. hypogaea) and its potential for introgression of wild genes into the peanut crop using induced allotetraploids"

Article Title: Arachis batizocoi: a study of its relationship to cultivated peanut (A. hypogaea) and its potential for introgression of wild genes into the peanut crop using induced allotetraploids

Journal: Annals of Botany

doi: 10.1093/aob/mcu237

Cytogenetic analysis of the hybrid between A. hypogaea ‘55437’ and ( A. batizocoi K9484 × A. stenosperna V10309) using GISH. Metaphase spreads of the hybrid were probed with a mixture of dig-labelled total genomic DNA from A. duranensis V14167 and biotin-labelled total genomic DNA from A. ipaënsis . (A) DAPI counterstain showing 40 chromosomes, 30 of which had strong centromeric DAPI bands, typical for Arachis A genome and A. batizocoi chromosomes. The coloured arrows indicate the nature of the individual chromosomes as derived from the combination of DAPI banding and GISH results (see text): green arrows, A. duranensis or A. stenosperma ; red arrows, A. ipaënsis ; orange arrows, A. batizocoi . (B) Superimposed image made from the individual FITC and Cy3 channels after GISH showing 20 green chromosomes that hybridized with the A. duranensis A genome probe and 20 red-orange chromosomes from hybridization with the A. ipaensis B genome probe. The circle comprises nine red-orange chromosomes, comprised of four A. ipaënsis and five A. batizocoi chromosomes. Comparison of hybridization intensities shows that the A. ipaënsis probe hybridized slightly more weakly to the A. batizocoi chromosomes.
Figure Legend Snippet: Cytogenetic analysis of the hybrid between A. hypogaea ‘55437’ and ( A. batizocoi K9484 × A. stenosperna V10309) using GISH. Metaphase spreads of the hybrid were probed with a mixture of dig-labelled total genomic DNA from A. duranensis V14167 and biotin-labelled total genomic DNA from A. ipaënsis . (A) DAPI counterstain showing 40 chromosomes, 30 of which had strong centromeric DAPI bands, typical for Arachis A genome and A. batizocoi chromosomes. The coloured arrows indicate the nature of the individual chromosomes as derived from the combination of DAPI banding and GISH results (see text): green arrows, A. duranensis or A. stenosperma ; red arrows, A. ipaënsis ; orange arrows, A. batizocoi . (B) Superimposed image made from the individual FITC and Cy3 channels after GISH showing 20 green chromosomes that hybridized with the A. duranensis A genome probe and 20 red-orange chromosomes from hybridization with the A. ipaensis B genome probe. The circle comprises nine red-orange chromosomes, comprised of four A. ipaënsis and five A. batizocoi chromosomes. Comparison of hybridization intensities shows that the A. ipaënsis probe hybridized slightly more weakly to the A. batizocoi chromosomes.

Techniques Used: Derivative Assay, Hybridization

34) Product Images from "HIC1 Expression Distinguishes Intestinal Carcinomas Sensitive to Chemotherapy"

Article Title: HIC1 Expression Distinguishes Intestinal Carcinomas Sensitive to Chemotherapy

Journal: Translational Oncology

doi: 10.1016/j.tranon.2016.01.005

Identification of the CIMP-related and gene expression–related HIC1 methylation sites. Top, the HIC1 gene exon-intron structure indicating the position of the major HIC1a transcript driven from the P1 promoter; the position of two alternative promoters P2 and P0 is also shown. A color-based classification of two CpG islands and other elements is indicated under the schemes; methylation probes covering the HIC1 locus are indicated as horizontal bars. The bottom panels depict the HIC1 methylation status in 277 CRC cases present in the TCGA 450K dataset. In the left panel, the specimens were grouped according to the CIMP status of tumor DNA; each specimen is represented by a horizontal bar. The right panel shows the HIC1 methylation status in CRCs classified as HIC1-high or HIC1-low; the sample with the HIC1 locus deletion is indicated by red arrow. The extent of methylation of CIMP-related (yellow) and expression-related probes (green) is shown as the gray-shaded heatmap of beta values (unmethylated: white; completely methylated: black); notice the different “slope” of beta values between the left and right panel. Probes are listed vertically with their identification code in a successive order according to their target sequence coordinates (the 5′ gene end is shown on the left). The CRC methylation profile of four probes boxed in pink is shown in Supplementary Figure S1. The correlation between HIC1 expression and the methylation profile of these four probes is given in Supplementary Figure S2.
Figure Legend Snippet: Identification of the CIMP-related and gene expression–related HIC1 methylation sites. Top, the HIC1 gene exon-intron structure indicating the position of the major HIC1a transcript driven from the P1 promoter; the position of two alternative promoters P2 and P0 is also shown. A color-based classification of two CpG islands and other elements is indicated under the schemes; methylation probes covering the HIC1 locus are indicated as horizontal bars. The bottom panels depict the HIC1 methylation status in 277 CRC cases present in the TCGA 450K dataset. In the left panel, the specimens were grouped according to the CIMP status of tumor DNA; each specimen is represented by a horizontal bar. The right panel shows the HIC1 methylation status in CRCs classified as HIC1-high or HIC1-low; the sample with the HIC1 locus deletion is indicated by red arrow. The extent of methylation of CIMP-related (yellow) and expression-related probes (green) is shown as the gray-shaded heatmap of beta values (unmethylated: white; completely methylated: black); notice the different “slope” of beta values between the left and right panel. Probes are listed vertically with their identification code in a successive order according to their target sequence coordinates (the 5′ gene end is shown on the left). The CRC methylation profile of four probes boxed in pink is shown in Supplementary Figure S1. The correlation between HIC1 expression and the methylation profile of these four probes is given in Supplementary Figure S2.

Techniques Used: Expressing, Methylation, Sequencing

Analysis of HIC1 expression in colorectal neoplasia. (A) HIC1 expression changes during tumor progression. qRT-PCR analysis of the HIC1 mRNA levels in healthy tissue, hyperplastic adenomas (Hyp; n = 9), adenomas displaying low-grade (LGD; n = 24) or high-grade (HGD; n = 25) dysplasia, and CRC ( n = 12). The boxed areas correspond to the second and third quartiles; the median of ΔCp values for each category is indicated as the red line. The relation between the HIC1 expression profile and neoplasia progression is significant, as evidenced by the Spearman’s ( ρ = − 0.67) and Kendall’s ( τ = − 0.51) coefficient values. (B) HIC1 expression in CRC subgroups clustered according to the DNA methylation profiles. HIC1-high and HIC1-low samples are indicated by brown or blue dots, respectively. Black dots indicate the other specimens. The red lines correspond to the median values; log2-expression intensity, binary logarithm of expression intensity (additional details are given in Supplementary Methods).
Figure Legend Snippet: Analysis of HIC1 expression in colorectal neoplasia. (A) HIC1 expression changes during tumor progression. qRT-PCR analysis of the HIC1 mRNA levels in healthy tissue, hyperplastic adenomas (Hyp; n = 9), adenomas displaying low-grade (LGD; n = 24) or high-grade (HGD; n = 25) dysplasia, and CRC ( n = 12). The boxed areas correspond to the second and third quartiles; the median of ΔCp values for each category is indicated as the red line. The relation between the HIC1 expression profile and neoplasia progression is significant, as evidenced by the Spearman’s ( ρ = − 0.67) and Kendall’s ( τ = − 0.51) coefficient values. (B) HIC1 expression in CRC subgroups clustered according to the DNA methylation profiles. HIC1-high and HIC1-low samples are indicated by brown or blue dots, respectively. Black dots indicate the other specimens. The red lines correspond to the median values; log2-expression intensity, binary logarithm of expression intensity (additional details are given in Supplementary Methods).

Techniques Used: Expressing, Quantitative RT-PCR, DNA Methylation Assay

Cytosine methylation analysis of the HIC1 locus. (A) Original and bisulfite-modified DNA sequence containing the second (alternative) HIC1 exon (boxed in grey). Methylation probes included in the region are indicated by red arrows above the sequences. CpG dinucleotides located in the sequence covered by the probes are in red; other CpG dinucleotides are in green. (B) Methylation of genomic DNA isolated from colorectal polyps displaying HGD and genomic DNA obtained from matched healthy tissue. Open circles mark unmethylated CpG dinucleotides, whereas closed circles indicate methylated CpGs; the numbering and color code of CpGs correspond to their positions in the analyzed genomic DNA as indicated in (A). (C) Cytosine methylation analysis of genomic DNA isolated from three HIC1-high and three HIC1-low CRCs.
Figure Legend Snippet: Cytosine methylation analysis of the HIC1 locus. (A) Original and bisulfite-modified DNA sequence containing the second (alternative) HIC1 exon (boxed in grey). Methylation probes included in the region are indicated by red arrows above the sequences. CpG dinucleotides located in the sequence covered by the probes are in red; other CpG dinucleotides are in green. (B) Methylation of genomic DNA isolated from colorectal polyps displaying HGD and genomic DNA obtained from matched healthy tissue. Open circles mark unmethylated CpG dinucleotides, whereas closed circles indicate methylated CpGs; the numbering and color code of CpGs correspond to their positions in the analyzed genomic DNA as indicated in (A). (C) Cytosine methylation analysis of genomic DNA isolated from three HIC1-high and three HIC1-low CRCs.

Techniques Used: Methylation, Modification, Sequencing, Isolation

35) Product Images from "Low Percentage of KRAS Mutations Revealed by Locked Nucleic Acid Polymerase Chain Reaction: Implications for Treatment of Metastatic Colorectal Cancer"

Article Title: Low Percentage of KRAS Mutations Revealed by Locked Nucleic Acid Polymerase Chain Reaction: Implications for Treatment of Metastatic Colorectal Cancer

Journal: Molecular Medicine

doi: 10.2119/molmed.2012.00175

Sensitivity threshold of PCR/sequencing, TheraScreen and LNA-PCR assays. DNA from HCT 116 cells carrying the gac codon 13 KRAS mutation (G13D) was diluted with the KRAS wild-type DNA from Colo 320 cells in proportions ranging from 50% to 0.125% mutant
Figure Legend Snippet: Sensitivity threshold of PCR/sequencing, TheraScreen and LNA-PCR assays. DNA from HCT 116 cells carrying the gac codon 13 KRAS mutation (G13D) was diluted with the KRAS wild-type DNA from Colo 320 cells in proportions ranging from 50% to 0.125% mutant

Techniques Used: Polymerase Chain Reaction, Sequencing, Mutagenesis

36) Product Images from "Whole DNA methylome profiling in mice exposed to secondhand smoke"

Article Title: Whole DNA methylome profiling in mice exposed to secondhand smoke

Journal: Epigenetics

doi: 10.4161/epi.22453

Figure 1. Genome-wide profiling of DNA methylation in SHS-exposed mice vs. control. The Hierarchical Clustering Analysis was used to generate heatmaps of DNA methylation profile in the lung of SHS-exposed mice and control. Representative heatmaps
Figure Legend Snippet: Figure 1. Genome-wide profiling of DNA methylation in SHS-exposed mice vs. control. The Hierarchical Clustering Analysis was used to generate heatmaps of DNA methylation profile in the lung of SHS-exposed mice and control. Representative heatmaps

Techniques Used: Genome Wide, DNA Methylation Assay, Mouse Assay

Figure 5. Methylation profiling of SINE B1 repetitive DNA elements in SHS-exposed mice vs. control. Bisulfite sequencing of SINE B1 elements was performed on genomic DNA isolated from the lung of SHS-exposed and control mice using a published
Figure Legend Snippet: Figure 5. Methylation profiling of SINE B1 repetitive DNA elements in SHS-exposed mice vs. control. Bisulfite sequencing of SINE B1 elements was performed on genomic DNA isolated from the lung of SHS-exposed and control mice using a published

Techniques Used: Methylation, Mouse Assay, Methylation Sequencing, Isolation

Figure 4. Methylation profiling of IAP-LTR repetitive DNA elements in SHS-exposed mice vs. control. Bisulfite sequencing of IAP-LTR elements was performed on genomic DNA isolated from the lung of SHS-exposed and control mice using a published
Figure Legend Snippet: Figure 4. Methylation profiling of IAP-LTR repetitive DNA elements in SHS-exposed mice vs. control. Bisulfite sequencing of IAP-LTR elements was performed on genomic DNA isolated from the lung of SHS-exposed and control mice using a published

Techniques Used: Methylation, Mouse Assay, Methylation Sequencing, Isolation

Figure 2. Verification of marginal hypermethylation in the Cacna1 gene and hypomethylation in the H2-T23 gene in SHS-exposed mice vs. control. Genomic DNA isolated from the lung of SHS-exposed and control mice was treated with sodium bisulfite,
Figure Legend Snippet: Figure 2. Verification of marginal hypermethylation in the Cacna1 gene and hypomethylation in the H2-T23 gene in SHS-exposed mice vs. control. Genomic DNA isolated from the lung of SHS-exposed and control mice was treated with sodium bisulfite,

Techniques Used: Mouse Assay, Isolation

Figure 3. Methylation profiling of LINE L1 repetitive DNA elements in SHS-exposed mice vs. control. Bisulfite sequencing of LINE L1 elements was performed on genomic DNA isolated from the lung of SHS-exposed and control mice using a published
Figure Legend Snippet: Figure 3. Methylation profiling of LINE L1 repetitive DNA elements in SHS-exposed mice vs. control. Bisulfite sequencing of LINE L1 elements was performed on genomic DNA isolated from the lung of SHS-exposed and control mice using a published

Techniques Used: Methylation, Mouse Assay, Methylation Sequencing, Isolation

37) Product Images from "Identification of a Halogenase Involved in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius"

Article Title: Identification of a Halogenase Involved in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01209-16

(A) Structure of the ochratoxin A cluster in Aspergillus carbonarius . (B) Map of the AcOTAhal locus in Δ kusA and Δ AcOTAhal strains of A. carbonarius . (C) Southern blot hybridizations from genomic DNA digested with SalI. The restriction
Figure Legend Snippet: (A) Structure of the ochratoxin A cluster in Aspergillus carbonarius . (B) Map of the AcOTAhal locus in Δ kusA and Δ AcOTAhal strains of A. carbonarius . (C) Southern blot hybridizations from genomic DNA digested with SalI. The restriction

Techniques Used: Southern Blot

38) Product Images from "Improvement of Soybean Agrobacterium-Mediated Transformation Efficiency by Adding Glutamine and Asparagine into the Culture Media"

Article Title: Improvement of Soybean Agrobacterium-Mediated Transformation Efficiency by Adding Glutamine and Asparagine into the Culture Media

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19103039

Southern blot analysis of T1 transgenic plants. Plasmid and genomic DNA were digested with HindIII, and hybridized with the bar probe labeled with DIG. M: marker; P: plasmid DNA of pWMB123; L1-1: DNA from T1 plant with bar + from transgenic line 1; L7-5: DNA from T1 plant with bar + from transgenic line 7; L17-2 and L17-9: DNA from T1 plant with bar + from transgenic line 17. WT: DNA from nontransgenic plants.
Figure Legend Snippet: Southern blot analysis of T1 transgenic plants. Plasmid and genomic DNA were digested with HindIII, and hybridized with the bar probe labeled with DIG. M: marker; P: plasmid DNA of pWMB123; L1-1: DNA from T1 plant with bar + from transgenic line 1; L7-5: DNA from T1 plant with bar + from transgenic line 7; L17-2 and L17-9: DNA from T1 plant with bar + from transgenic line 17. WT: DNA from nontransgenic plants.

Techniques Used: Southern Blot, Transgenic Assay, Plasmid Preparation, Labeling, Marker

39) Product Images from "Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding"

Article Title: Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding

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

doi: 10.1073/pnas.1503157112

TER1 overexpression does not cause short telomeres. ( A ) Southern blots. Genomic DNA was extracted from cells harboring pSP1 and ter1 /pSP1 after seven successive streaks. DNA was digested with ApaI and hybridized to a telomeric probe. Molecular weight
Figure Legend Snippet: TER1 overexpression does not cause short telomeres. ( A ) Southern blots. Genomic DNA was extracted from cells harboring pSP1 and ter1 /pSP1 after seven successive streaks. DNA was digested with ApaI and hybridized to a telomeric probe. Molecular weight

Techniques Used: Over Expression, Molecular Weight

Telomere lengths in heterozygous ter1 diploid cells. Shown is the telomere blot. Genomic DNA from heterozygous diploid ter1 +/MUTANT cells was isolated after cells were streaked > 5 times. DNA was digested with ApaI and hybridized to a telomeric
Figure Legend Snippet: Telomere lengths in heterozygous ter1 diploid cells. Shown is the telomere blot. Genomic DNA from heterozygous diploid ter1 +/MUTANT cells was isolated after cells were streaked > 5 times. DNA was digested with ApaI and hybridized to a telomeric

Techniques Used: Mutagenesis, Isolation

Telomeric DNA from ter1 -STE loop cells has increased rare cytosines and reduced guanine tracts. ( A ) Telomere blot of genomic DNA was extracted from wild-type and ter1 -STE loop cells after three successive streaks at 37 °C and one subsequent streak
Figure Legend Snippet: Telomeric DNA from ter1 -STE loop cells has increased rare cytosines and reduced guanine tracts. ( A ) Telomere blot of genomic DNA was extracted from wild-type and ter1 -STE loop cells after three successive streaks at 37 °C and one subsequent streak

Techniques Used:

40) Product Images from "Comparative Genomic In Situ Hybridization and the Possible Role of Retroelements in the Karyotypic Evolution of Three Akodontini Species"

Article Title: Comparative Genomic In Situ Hybridization and the Possible Role of Retroelements in the Karyotypic Evolution of Three Akodontini Species

Journal: International Journal of Genomics

doi: 10.1155/2017/5935380

Interspecific GISH among Akodon cursor (ACU), A. montensis (AMO), and Necromys lasiurus (NLA). The initials on the left correspond to the species cells. The labeled DNA used is identified in green, and the suppressor DNA is represented in white. All the cells were counterstained with propidium iodide. Bar = 10 μ m.
Figure Legend Snippet: Interspecific GISH among Akodon cursor (ACU), A. montensis (AMO), and Necromys lasiurus (NLA). The initials on the left correspond to the species cells. The labeled DNA used is identified in green, and the suppressor DNA is represented in white. All the cells were counterstained with propidium iodide. Bar = 10 μ m.

Techniques Used: Labeling

Interspecific GISH among Akodon cursor (ACU) and A. montensis (AMO). The initials on the left correspond to the species cells. The labeled DNA used is identified in green and the suppressor DNA is represented in white. All the cells were counterstained with propidium iodide. Bar = 10 μ m.
Figure Legend Snippet: Interspecific GISH among Akodon cursor (ACU) and A. montensis (AMO). The initials on the left correspond to the species cells. The labeled DNA used is identified in green and the suppressor DNA is represented in white. All the cells were counterstained with propidium iodide. Bar = 10 μ m.

Techniques Used: Labeling

41) Product Images from "Coupling transcriptional activation of CRISPR–Cas system and DNA repair genes by Csa3a in Sulfolobus islandicus"

Article Title: Coupling transcriptional activation of CRISPR–Cas system and DNA repair genes by Csa3a in Sulfolobus islandicus

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx612

Csa3a binding to the leader sequence and activating CRISPR transcription. ( A ) CRISPR RNA activation by Csa3a revealed by transcriptome data. Black arrows: up-regulated CRISPR RNAs in Δ cas1 :: csa3a and Δ cas3cmr2αcmr2β :: csa3a strains; red arrows: up-regulated CRISPR RNAs in wt:: csa3a , Δ cas1 :: csa3a and Δ cas3cmr2αcmr2β :: csa3a strains, compared with their respective control strains. ( B ) DNA motifs in leader sequences and probes used for EMSA experiments. Two DNA motifs in leader sequences similar to the Csa3a-binding site are shown in dark and light blue, and their sequences are shown above or below the motifs. A putative TATA box is shown within the red rectangle. Wt and mutation DNA probes for EMSA experiments and their nucleotide ranges relative to the first repeat are indicated. ( C ) EMSA experiments with increasing amount of Csa3a (10, 20 and 40 ng/μl; 10 ng/μl = 0.4 pmol/μl) or unlabelled cold probe (6- or 12-fold excess) using a biotin-labeled full-leader sequence as the probe (10 ng/μl). ( D ) EMSA experiments with increasing amounts of Csa3a (10, 20 and 40 ng/μl) or unlabelled cold probe (2-, 6- or 12-fold excess) using HEX-labeled Probe 2 (10 ng/μl). E, EMSA experiments performed with increasing amounts of Csa3a (10, 20 and 40 ng/μl), using HEX-labelled Probe 3 (10 ng/μl). ( F ) EMSA experiments performed with increasing amounts of Csa3a (10, 20 and 40 ng/μl), using HEX-labeled Probe 2 mut (P2mut) with mutation at the putative Csa3a-binding motif (10 ng/μl). The reaction with 40 ng/μL Csa3a and 10 ng/μl Probe 2 was used as the positive control.
Figure Legend Snippet: Csa3a binding to the leader sequence and activating CRISPR transcription. ( A ) CRISPR RNA activation by Csa3a revealed by transcriptome data. Black arrows: up-regulated CRISPR RNAs in Δ cas1 :: csa3a and Δ cas3cmr2αcmr2β :: csa3a strains; red arrows: up-regulated CRISPR RNAs in wt:: csa3a , Δ cas1 :: csa3a and Δ cas3cmr2αcmr2β :: csa3a strains, compared with their respective control strains. ( B ) DNA motifs in leader sequences and probes used for EMSA experiments. Two DNA motifs in leader sequences similar to the Csa3a-binding site are shown in dark and light blue, and their sequences are shown above or below the motifs. A putative TATA box is shown within the red rectangle. Wt and mutation DNA probes for EMSA experiments and their nucleotide ranges relative to the first repeat are indicated. ( C ) EMSA experiments with increasing amount of Csa3a (10, 20 and 40 ng/μl; 10 ng/μl = 0.4 pmol/μl) or unlabelled cold probe (6- or 12-fold excess) using a biotin-labeled full-leader sequence as the probe (10 ng/μl). ( D ) EMSA experiments with increasing amounts of Csa3a (10, 20 and 40 ng/μl) or unlabelled cold probe (2-, 6- or 12-fold excess) using HEX-labeled Probe 2 (10 ng/μl). E, EMSA experiments performed with increasing amounts of Csa3a (10, 20 and 40 ng/μl), using HEX-labelled Probe 3 (10 ng/μl). ( F ) EMSA experiments performed with increasing amounts of Csa3a (10, 20 and 40 ng/μl), using HEX-labeled Probe 2 mut (P2mut) with mutation at the putative Csa3a-binding motif (10 ng/μl). The reaction with 40 ng/μL Csa3a and 10 ng/μl Probe 2 was used as the positive control.

Techniques Used: Binding Assay, Sequencing, CRISPR, Activation Assay, Mutagenesis, Labeling, Positive Control

42) Product Images from "Laser capture microdissection and metagenomic analysis of intact mucosa-associated microbial communities of human colon"

Article Title: Laser capture microdissection and metagenomic analysis of intact mucosa-associated microbial communities of human colon

Journal: Applied microbiology and biotechnology

doi: 10.1007/s00253-010-2921-8

Optimization of phi29 multiple displacement amplification ( MDA ) with complex DNA templates. a 16S rRNA gene PCR and T-RFLP analysis of fidelity of MDA with the different amount of DNA templates. High fidelity of MDA is observed in the reactions using
Figure Legend Snippet: Optimization of phi29 multiple displacement amplification ( MDA ) with complex DNA templates. a 16S rRNA gene PCR and T-RFLP analysis of fidelity of MDA with the different amount of DNA templates. High fidelity of MDA is observed in the reactions using

Techniques Used: Multiple Displacement Amplification, Polymerase Chain Reaction

Overview of fidelity of phi29 MDA on laser capture micro-dissected DNA samples using 16S rRNA gene library cloning and sequencing. Bacterial composition is compared before and after phi29 MDA, based on the alignment with RDP. Operational taxonomic units
Figure Legend Snippet: Overview of fidelity of phi29 MDA on laser capture micro-dissected DNA samples using 16S rRNA gene library cloning and sequencing. Bacterial composition is compared before and after phi29 MDA, based on the alignment with RDP. Operational taxonomic units

Techniques Used: Multiple Displacement Amplification, Clone Assay, Sequencing

Distribution of classified metagenomic-derived sequences obtained from human colon samples. Genomic DNA was isolated from the mucosa of biopsy samples and subjected to phi29 polymerase multiple displacement amplification ( MDA ). Note the difference in
Figure Legend Snippet: Distribution of classified metagenomic-derived sequences obtained from human colon samples. Genomic DNA was isolated from the mucosa of biopsy samples and subjected to phi29 polymerase multiple displacement amplification ( MDA ). Note the difference in

Techniques Used: Derivative Assay, Isolation, Multiple Displacement Amplification

43) Product Images from "RNA‐guided Cas9 as an in vivo desired‐target mutator in maize"

Article Title: RNA‐guided Cas9 as an in vivo desired‐target mutator in maize

Journal: Plant Biotechnology Journal

doi: 10.1111/pbi.12739

Editing of the LG 1 gene to confer upright leaves and a compact maize plant architecture. (a) Construction of the expression cassette for RNA ‐guided Cas9 targeted genes. (b) The sg RNA mediating site is indicated in exon 1 within the gene structure of Zm LG 1 . The Sfc I restriction enzyme recognition sequence was selected within the designed mutated region of the Cas9 nuclease. The sequence of the single guiding RNA (sg RNA ) region is shaded in grey. (c) PCR ‐ RE ( Sfc I) assay (marker) profiles for 15 randomly selected T0 sample plants. The wild‐type sequence should be cut into two bands ( WT cut). In samples 1, 2, 3, 5, 9, 10, 14 and 15, both copies of LG 1 were mutated were not cleaved. Samples 4 and 8 harboured the wild‐type allele. Samples 6, 7, 11, 12 and 13 were heterozygous for the wild‐type and targeted mutant genotypes ( LG 1 , lg1 ). M, Tiangen D2000 2 K DNA marker (Tiangen, Beijing, China). (d) Relatively high frequency of targeted mutation events ( n > 5) among 207 editing events. (e) Phenotype of a 2‐week‐old wild‐type ZC 01 seedling ( LG 1 , LG 1 ); (f). Phenotype of the generated LG 1 ‐null mutation seedling with the same genetic background as the wild‐type seedling, ZC 01 ( lg1 , lg1 ). (g–j). SEM of the adaxial (g, h) and abaxial (i, j) surfaces of the junction of the sheath and leaf of the 2‐week‐old plant showing the ligule and auricle phenotypes. The wild‐type plant (g and i) exhibited a ligule and auricle, but the generated mutant (h and j) lacked both a ligule and auricle. The upright leaves and compact plant architecture were evident throughout the growing stage until the late stage (k), due to the flat angle between the sheath and leaves that resulted from ligule and auricle mutation (l).
Figure Legend Snippet: Editing of the LG 1 gene to confer upright leaves and a compact maize plant architecture. (a) Construction of the expression cassette for RNA ‐guided Cas9 targeted genes. (b) The sg RNA mediating site is indicated in exon 1 within the gene structure of Zm LG 1 . The Sfc I restriction enzyme recognition sequence was selected within the designed mutated region of the Cas9 nuclease. The sequence of the single guiding RNA (sg RNA ) region is shaded in grey. (c) PCR ‐ RE ( Sfc I) assay (marker) profiles for 15 randomly selected T0 sample plants. The wild‐type sequence should be cut into two bands ( WT cut). In samples 1, 2, 3, 5, 9, 10, 14 and 15, both copies of LG 1 were mutated were not cleaved. Samples 4 and 8 harboured the wild‐type allele. Samples 6, 7, 11, 12 and 13 were heterozygous for the wild‐type and targeted mutant genotypes ( LG 1 , lg1 ). M, Tiangen D2000 2 K DNA marker (Tiangen, Beijing, China). (d) Relatively high frequency of targeted mutation events ( n > 5) among 207 editing events. (e) Phenotype of a 2‐week‐old wild‐type ZC 01 seedling ( LG 1 , LG 1 ); (f). Phenotype of the generated LG 1 ‐null mutation seedling with the same genetic background as the wild‐type seedling, ZC 01 ( lg1 , lg1 ). (g–j). SEM of the adaxial (g, h) and abaxial (i, j) surfaces of the junction of the sheath and leaf of the 2‐week‐old plant showing the ligule and auricle phenotypes. The wild‐type plant (g and i) exhibited a ligule and auricle, but the generated mutant (h and j) lacked both a ligule and auricle. The upright leaves and compact plant architecture were evident throughout the growing stage until the late stage (k), due to the flat angle between the sheath and leaves that resulted from ligule and auricle mutation (l).

Techniques Used: Expressing, Sequencing, Polymerase Chain Reaction, Marker, Mutagenesis, Generated

44) 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

45) Product Images from "Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes"

Article Title: Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1038/mtna.2016.19

Design and activity of transcription activator-like nucleases (TALENs) targeted to exon 80 and intron 79. Three pairs of TALENs with targets across intron 79 and exon 80 ( a ) were expressed with adenoviral vectors in RDEB-E67A6 keratinocytes. A polymerase chain reaction (PCR) product spanning the TALEN target sites was generated with primers F1/R and analyzed with the Cel I (Surveyor) mutation detection assay, showing efficient indel generation activity for the T6/T7 pair, only weak activity for T14/T15 and no detectable activity for T1/T2. Solid arrowhead indicates uncleaved DNA, arrowheads indicate cleavage fragments. Percentage of cleavage for each PCR product is shown at the bottom. IX molecular weight marker (MW) ( b ). Schematic drawing showing the position of T6 and T7 TALENs on a genomic DNA sequence fragment containing COL7A1 exon 80 (blue) and the c.6527insC mutation site (red arrowhead). TALENs spacer sequence is shaded ( c ). RDEB, recessive dystrophic epidermolysis bullosa.
Figure Legend Snippet: Design and activity of transcription activator-like nucleases (TALENs) targeted to exon 80 and intron 79. Three pairs of TALENs with targets across intron 79 and exon 80 ( a ) were expressed with adenoviral vectors in RDEB-E67A6 keratinocytes. A polymerase chain reaction (PCR) product spanning the TALEN target sites was generated with primers F1/R and analyzed with the Cel I (Surveyor) mutation detection assay, showing efficient indel generation activity for the T6/T7 pair, only weak activity for T14/T15 and no detectable activity for T1/T2. Solid arrowhead indicates uncleaved DNA, arrowheads indicate cleavage fragments. Percentage of cleavage for each PCR product is shown at the bottom. IX molecular weight marker (MW) ( b ). Schematic drawing showing the position of T6 and T7 TALENs on a genomic DNA sequence fragment containing COL7A1 exon 80 (blue) and the c.6527insC mutation site (red arrowhead). TALENs spacer sequence is shaded ( c ). RDEB, recessive dystrophic epidermolysis bullosa.

Techniques Used: Activity Assay, TALENs, Polymerase Chain Reaction, Generated, Mutagenesis, Detection Assay, Molecular Weight, Marker, Sequencing

46) Product Images from "Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells"

Article Title: Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005872

The gene structure of the piggyBac transformation vector, pBac[pAAPP-mBax; 3xP3-EGFP], TG mosquito lines, and insertion sites. (A) The gene construct derived from the piggyBac -based vector contains piggyBac Left-arm (L) and Right-arm (R) with an inverted terminal repeat (ITR). The T7-mBax gene is expressed under the control of the An . stephensi aapp promoter (pAAPP) and An . gambiae trypsin terminator (Tryter). The transformation marker, EGFP is expressed under the control of the 3xP3 promoter. A double line represents the probe region for a Southern blot analysis. The restriction enzyme ( Msp I) site is represented below the scheme. The red arrow represents the primer sites for a RT-PCR analysis in Fig 2 . (B) A Southern blot analysis of AAPP-mBax lines. Genomic DNA from AAPP-mBax mosquito lines (lines 1 and 3) was digested with Msp I, and hybridized with a fragment corresponding to the piggyBac R region. (C) Insertion sites of the transgene in AAPP-mBax lines 1 and 3. The blue bars show the local DNA region within each genomic scaffold. Black boxes represent the annotated protein-coding region in the VectorBase ( https://www.vectorbase.org/ ). Double-headed arrows show the piggyBac construct. L: piggyBac Left-arm, R: piggyBac Right-arm.
Figure Legend Snippet: The gene structure of the piggyBac transformation vector, pBac[pAAPP-mBax; 3xP3-EGFP], TG mosquito lines, and insertion sites. (A) The gene construct derived from the piggyBac -based vector contains piggyBac Left-arm (L) and Right-arm (R) with an inverted terminal repeat (ITR). The T7-mBax gene is expressed under the control of the An . stephensi aapp promoter (pAAPP) and An . gambiae trypsin terminator (Tryter). The transformation marker, EGFP is expressed under the control of the 3xP3 promoter. A double line represents the probe region for a Southern blot analysis. The restriction enzyme ( Msp I) site is represented below the scheme. The red arrow represents the primer sites for a RT-PCR analysis in Fig 2 . (B) A Southern blot analysis of AAPP-mBax lines. Genomic DNA from AAPP-mBax mosquito lines (lines 1 and 3) was digested with Msp I, and hybridized with a fragment corresponding to the piggyBac R region. (C) Insertion sites of the transgene in AAPP-mBax lines 1 and 3. The blue bars show the local DNA region within each genomic scaffold. Black boxes represent the annotated protein-coding region in the VectorBase ( https://www.vectorbase.org/ ). Double-headed arrows show the piggyBac construct. L: piggyBac Left-arm, R: piggyBac Right-arm.

Techniques Used: Transformation Assay, Plasmid Preparation, Construct, Derivative Assay, Marker, Southern Blot, Reverse Transcription Polymerase Chain Reaction

47) Product Images from "A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants"

Article Title: A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038718

Southern blot analysis of transgenic tobacco plants. (A) HTG7; (B) AM79; (C) A1501; (D) RD; (E) G2. Five transgenic lines each construct were analyzed. 100 µg genomic DNA was digested with Hin dIII which has only one site in the plasmid, so the band numbers are equal to the copy number of transgene.
Figure Legend Snippet: Southern blot analysis of transgenic tobacco plants. (A) HTG7; (B) AM79; (C) A1501; (D) RD; (E) G2. Five transgenic lines each construct were analyzed. 100 µg genomic DNA was digested with Hin dIII which has only one site in the plasmid, so the band numbers are equal to the copy number of transgene.

Techniques Used: Southern Blot, Transgenic Assay, Construct, Plasmid Preparation

48) Product Images from "Differential Expression of Non-Coding RNAs and Continuous Evolution of the X Chromosome in Testicular Transcriptome of Two Mouse Species"

Article Title: Differential Expression of Non-Coding RNAs and Continuous Evolution of the X Chromosome in Testicular Transcriptome of Two Mouse Species

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017198

Quantitative real-time RT-PCR of piRNA clusters from Chromosome 2. (a) Two nested primer pairs were designed (outer and inner primer pairs), which amplify parts of all four piRNA clusters in chromosomal region chr2: 150,953,000–151,257,000. (b) Detailed view of part of the first cluster, which shows localization of the outer and inner primer PCR products. (c) Quantitative real-time PCR of testicular DNA indicate different copy number between Spr and B6. (d) Strong downregulation was observed in testicular cDNA. (e) In B6 the piRNA clusters were predominantly expressed in pachytene spermatocytes, with substantial downregulation in Spr. Two or three biological samples were used for each comparison. The expression was related to concentration and normalized to B6 expression. Standard deviations are plotted. Pre-pach: pre-pachytene spermatocytes; pach SC: pachytene spermatocytes; ST: spermatids.
Figure Legend Snippet: Quantitative real-time RT-PCR of piRNA clusters from Chromosome 2. (a) Two nested primer pairs were designed (outer and inner primer pairs), which amplify parts of all four piRNA clusters in chromosomal region chr2: 150,953,000–151,257,000. (b) Detailed view of part of the first cluster, which shows localization of the outer and inner primer PCR products. (c) Quantitative real-time PCR of testicular DNA indicate different copy number between Spr and B6. (d) Strong downregulation was observed in testicular cDNA. (e) In B6 the piRNA clusters were predominantly expressed in pachytene spermatocytes, with substantial downregulation in Spr. Two or three biological samples were used for each comparison. The expression was related to concentration and normalized to B6 expression. Standard deviations are plotted. Pre-pach: pre-pachytene spermatocytes; pach SC: pachytene spermatocytes; ST: spermatids.

Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, SPR Assay, Expressing, Concentration Assay

49) Product Images from "Promoter Demethylation and Chromatin Remodeling by Green Tea Polyphenols Leads to Re-expression of GSTP1 in Human Prostate Cancer Cells"

Article Title: Promoter Demethylation and Chromatin Remodeling by Green Tea Polyphenols Leads to Re-expression of GSTP1 in Human Prostate Cancer Cells

Journal: International journal of cancer. Journal international du cancer

doi: 10.1002/ijc.24988

Effect of green tea polyphenols (GTP), its major constituent, epigallocatechin-3-gallate (EGCG) and nucleoside DNA methyltransferase inhibitor, 5-aza-2′deoxycitidine (5-Aza-dC) on glutathione-S-transferase pi (GSTP1) re-expression in human prostate cancer LNCaP cells. A, dose- and time- dependent re-expression of GSTP1 protein by 5–10μg/mL GTP, 5–10μM 5-Aza-dC and 10–20μM of EGCG by using ELISA assay from Biotrin International. The results are mean of 6–8 determinations and are analyzed by one way ANOVA, bars ± SD and ** P
Figure Legend Snippet: Effect of green tea polyphenols (GTP), its major constituent, epigallocatechin-3-gallate (EGCG) and nucleoside DNA methyltransferase inhibitor, 5-aza-2′deoxycitidine (5-Aza-dC) on glutathione-S-transferase pi (GSTP1) re-expression in human prostate cancer LNCaP cells. A, dose- and time- dependent re-expression of GSTP1 protein by 5–10μg/mL GTP, 5–10μM 5-Aza-dC and 10–20μM of EGCG by using ELISA assay from Biotrin International. The results are mean of 6–8 determinations and are analyzed by one way ANOVA, bars ± SD and ** P

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

Effect of green tea polyphenols (GTP) on reversal of methylation in human prostate cancer LNCaP cells. A, Map of the GSTP1 gene and putative CpG islands. The relative position and sizes of the 7 exons (solid bars) of the GSTP1 are depicted. B, MS-PCR for GSTP1 promoter on genomic DNA isolated from cells after treatment with 10μg/mL GTP for 7 days and 10μM 5-Aza-dC for 3 days. Displayed are the products generated with primers specific for unmethylated GSTP1 CpG island alleles (U) and for hypermethylated GSTP1 CpG island alleles (M). C, methylation analysis of individual clones from proximal and D, distal GSTP1 promoter after GTP and 5-Aza-dC treatment. Reduction in GSTP1 CpG island hypermethylation was noted in LNCaP DNA by these treatments. At least 15 PCR clones from each treatment were sequenced; the CpG island DNA methylation pattern for selected clones are displayed. Closed circles indicate a methylated CpG and open circles indicate an unmethylated CpG. The details are described in ‘Materials and Methods’ section.
Figure Legend Snippet: Effect of green tea polyphenols (GTP) on reversal of methylation in human prostate cancer LNCaP cells. A, Map of the GSTP1 gene and putative CpG islands. The relative position and sizes of the 7 exons (solid bars) of the GSTP1 are depicted. B, MS-PCR for GSTP1 promoter on genomic DNA isolated from cells after treatment with 10μg/mL GTP for 7 days and 10μM 5-Aza-dC for 3 days. Displayed are the products generated with primers specific for unmethylated GSTP1 CpG island alleles (U) and for hypermethylated GSTP1 CpG island alleles (M). C, methylation analysis of individual clones from proximal and D, distal GSTP1 promoter after GTP and 5-Aza-dC treatment. Reduction in GSTP1 CpG island hypermethylation was noted in LNCaP DNA by these treatments. At least 15 PCR clones from each treatment were sequenced; the CpG island DNA methylation pattern for selected clones are displayed. Closed circles indicate a methylated CpG and open circles indicate an unmethylated CpG. The details are described in ‘Materials and Methods’ section.

Techniques Used: Methylation, Mass Spectrometry, Polymerase Chain Reaction, Isolation, Generated, Clone Assay, DNA Methylation Assay

Effect of green tea polyphenols (GTP), its major constituent, epigallocatechin-3-gallate (EGCG) and nucleoside DNA methyltransferase inhibitor, 5-aza-2′deoxycitidine (5-Aza-dC) on 5-cytosine DNA methyltransferase (DNMT) in human prostate cancer LNCaP cells. A, dose-dependent inhibition of DNMT activity by various concentrations of GTP 5–20μg/mL and 5–20μM of EGCG and 5-Aza-dC B, time-dependent inhibition of DNMT activity at 10μg/mL GTP and 10μM of EGCG and 5-Aza-dC for 1–7 days. The results are mean of 6–8 determinations and are analyzed by one way ANOVA, bars ± SD. C, dose- and time- dependent inhibition of DNMT1 protein expression after GTP treatment, anti-Oct-1 is used as loading control, and D, dose- and time- dependent inhibition of DNMT1 mRNA by GTP at indicated doses and times. The results of mRNA levels are mean ± SD of 6–8 determinations ** P
Figure Legend Snippet: Effect of green tea polyphenols (GTP), its major constituent, epigallocatechin-3-gallate (EGCG) and nucleoside DNA methyltransferase inhibitor, 5-aza-2′deoxycitidine (5-Aza-dC) on 5-cytosine DNA methyltransferase (DNMT) in human prostate cancer LNCaP cells. A, dose-dependent inhibition of DNMT activity by various concentrations of GTP 5–20μg/mL and 5–20μM of EGCG and 5-Aza-dC B, time-dependent inhibition of DNMT activity at 10μg/mL GTP and 10μM of EGCG and 5-Aza-dC for 1–7 days. The results are mean of 6–8 determinations and are analyzed by one way ANOVA, bars ± SD. C, dose- and time- dependent inhibition of DNMT1 protein expression after GTP treatment, anti-Oct-1 is used as loading control, and D, dose- and time- dependent inhibition of DNMT1 mRNA by GTP at indicated doses and times. The results of mRNA levels are mean ± SD of 6–8 determinations ** P

Techniques Used: Inhibition, Activity Assay, Expressing

50) Product Images from "Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the 3D organisation of the A. thaliana genome"

Article Title: Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the 3D organisation of the A. thaliana genome

Journal: Cell reports

doi: 10.1016/j.celrep.2016.07.016

Identification of NADs in nuc1 mutant A. PCR detection of rRNA gene variant types in DNA of purified nuclei (N) or nucleoli (No) of wild-type (Col-0) or nuc1 plants. B. Chromosome plots displaying the relative enrichment of genomic sequence as 100kb windows in the nucleolus on each 5 chromosomes in nuc1 mutant. C. DAPI-stained nuclei from WT Col-0 (left panel) or from nuc1 plants (right panel). The green signal represent the fluorescence obtained from the anti-cenH3 antibody. Scale bar is 5μm. D. Box-plot showing the relative proportion of nucleolus-associated centromeres in WT Col-0 or in nuc1 mutant. The analysis was performed on 100 nuclei per samples. F. Box-plot displaying the relative number of heterochromatic signals inside the nucleolus in WT Col-0 or in nuc1 mutant. The asterisk is present when the enrichment is statistically demonstrated.
Figure Legend Snippet: Identification of NADs in nuc1 mutant A. PCR detection of rRNA gene variant types in DNA of purified nuclei (N) or nucleoli (No) of wild-type (Col-0) or nuc1 plants. B. Chromosome plots displaying the relative enrichment of genomic sequence as 100kb windows in the nucleolus on each 5 chromosomes in nuc1 mutant. C. DAPI-stained nuclei from WT Col-0 (left panel) or from nuc1 plants (right panel). The green signal represent the fluorescence obtained from the anti-cenH3 antibody. Scale bar is 5μm. D. Box-plot showing the relative proportion of nucleolus-associated centromeres in WT Col-0 or in nuc1 mutant. The analysis was performed on 100 nuclei per samples. F. Box-plot displaying the relative number of heterochromatic signals inside the nucleolus in WT Col-0 or in nuc1 mutant. The asterisk is present when the enrichment is statistically demonstrated.

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Variant Assay, Purification, Sequencing, Staining, Fluorescence

Telomere nucleolar clustering is affected in nuc1 mutant A. Chromosome-plot displaying the relative enrichment of genomic sequence as 100kb windows in the nucleolus on each 5 chromosomes of nuc1 mutant versus WT Col-0. B–C. DNA-FISH analyses using a telomere specific probe (red signal) on DAPI-stained nuclei (Blue) from WT Col-0 (B) or from nuc1 plants (C). D. Box-plot showing the proportion of nucleolus-associated telomeres in WT Col-0 (from 62 nuclei) and in nuc1 mutant (from 68 nuclei). The asterisk is present when the enrichment is statistically demonstrated.
Figure Legend Snippet: Telomere nucleolar clustering is affected in nuc1 mutant A. Chromosome-plot displaying the relative enrichment of genomic sequence as 100kb windows in the nucleolus on each 5 chromosomes of nuc1 mutant versus WT Col-0. B–C. DNA-FISH analyses using a telomere specific probe (red signal) on DAPI-stained nuclei (Blue) from WT Col-0 (B) or from nuc1 plants (C). D. Box-plot showing the proportion of nucleolus-associated telomeres in WT Col-0 (from 62 nuclei) and in nuc1 mutant (from 68 nuclei). The asterisk is present when the enrichment is statistically demonstrated.

Techniques Used: Mutagenesis, Sequencing, Fluorescence In Situ Hybridization, Staining

51) Product Images from "Mapping the Telomere Integrated Genome of Human Herpesvirus 6A and 6B"

Article Title: Mapping the Telomere Integrated Genome of Human Herpesvirus 6A and 6B

Journal: Virology

doi: 10.1016/j.virol.2013.03.030

Amplification and restriction enzyme digestion of DR L fragments from DNA isolated from PBMC of members of a family with inherited HHV-6
Figure Legend Snippet: Amplification and restriction enzyme digestion of DR L fragments from DNA isolated from PBMC of members of a family with inherited HHV-6

Techniques Used: Amplification, Isolation

Treatment of HHV-6A latently integrated HEK-293 cells with the histone deacetylase inhibitor TSA induces novel PCR products consistent with episome and/or concatemer formation and induction of lytic viral DNA replication
Figure Legend Snippet: Treatment of HHV-6A latently integrated HEK-293 cells with the histone deacetylase inhibitor TSA induces novel PCR products consistent with episome and/or concatemer formation and induction of lytic viral DNA replication

Techniques Used: Histone Deacetylase Assay, Polymerase Chain Reaction

52) Product Images from "DNA Fingerprinting Abnormalities Can Distinguish Ulcerative Colitis Patients with Dysplasia and Cancer from Those Who Are Dysplasia/Cancer-Free"

Article Title: DNA Fingerprinting Abnormalities Can Distinguish Ulcerative Colitis Patients with Dysplasia and Cancer from Those Who Are Dysplasia/Cancer-Free

Journal: The American Journal of Pathology

doi:

Cloning and characterization of a novel AP-PCR product band X. A: Fingerprint from AP-PCR. The arrow indicates the position where band X was located. B: The positions of primers and inverted Alu repeats on band X sequence. The dotted line represents the deleted sequence in high-grade dysplasia. C: Multiplex PCR of genomic DNA, the upper band indicates the presence of DNA with deletion, the lower bands indicate the presence of normal DNA. D: Multiplex PCR of microdissected DNA. NL, DNA from normal individual; F1 to F9, microdissected fractions 1 to 9.
Figure Legend Snippet: Cloning and characterization of a novel AP-PCR product band X. A: Fingerprint from AP-PCR. The arrow indicates the position where band X was located. B: The positions of primers and inverted Alu repeats on band X sequence. The dotted line represents the deleted sequence in high-grade dysplasia. C: Multiplex PCR of genomic DNA, the upper band indicates the presence of DNA with deletion, the lower bands indicate the presence of normal DNA. D: Multiplex PCR of microdissected DNA. NL, DNA from normal individual; F1 to F9, microdissected fractions 1 to 9.

Techniques Used: Clone Assay, Polymerase Chain Reaction, Sequencing, Multiplex Assay

53) Product Images from "Extensive epigenetic and transcriptomic variability between genetically identical human B-lymphoblastoid cells with implications in pharmacogenomics research"

Article Title: Extensive epigenetic and transcriptomic variability between genetically identical human B-lymphoblastoid cells with implications in pharmacogenomics research

Journal: Scientific Reports

doi: 10.1038/s41598-019-40897-9

Study design and basic characteristics of sGT LCLs. ( a ) Study design. ( b ) Capillary electropherograms of PCR-amplified STR regions. ( c ) The fraction of cells at each cell cycle stage based on DNA content and calculated DNA-indices (DIs).
Figure Legend Snippet: Study design and basic characteristics of sGT LCLs. ( a ) Study design. ( b ) Capillary electropherograms of PCR-amplified STR regions. ( c ) The fraction of cells at each cell cycle stage based on DNA content and calculated DNA-indices (DIs).

Techniques Used: Polymerase Chain Reaction, Amplification

54) Product Images from "Novel Isolation and Biochemical Characterization of Immortalized Fibroblasts for Tissue Engineering Vocal Fold Lamina Propria"

Article Title: Novel Isolation and Biochemical Characterization of Immortalized Fibroblasts for Tissue Engineering Vocal Fold Lamina Propria

Journal: Tissue Engineering. Part C, Methods

doi: 10.1089/ten.tec.2008.0390

Southern blot (telomere length assay) of transduced cell lines and parental hVFFs. Genomic DNA was isolated from hTERT -transduced populations, control, and uninfected parental lines at passages 8 and 18, and then hybridized with a telomeric probe to visualize
Figure Legend Snippet: Southern blot (telomere length assay) of transduced cell lines and parental hVFFs. Genomic DNA was isolated from hTERT -transduced populations, control, and uninfected parental lines at passages 8 and 18, and then hybridized with a telomeric probe to visualize

Techniques Used: Southern Blot, Isolation

55) Product Images from "Molecular Characterization of an Endopolygalacturonase from Fusarium oxysporum Expressed during Early Stages of Infection"

Article Title: Molecular Characterization of an Endopolygalacturonase from Fusarium oxysporum Expressed during Early Stages of Infection

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.67.5.2191-2196.2001

Southern hybridization analysis of RT-PCR products, showing the expression pattern of pg5 during infection of tomato plants by F. oxysporum f. sp. lycopersici . First-strand cDNAs were generated from total RNA isolated at the indicated time points (in hours) from roots and stems of uninfected or infected plants and used as templates for PCR with primers specific for pg5 (see Materials and Methods). Aliquots of the PCR products were electrophoresed on a 2% agarose gel, blotted onto a nylon membrane, and hybridized with the labeled pg5 probe. The position and size (in base pairs) of the pg5 fragment are indicated. The numbers represent days after inoculation. gDNA, genomic DNA.
Figure Legend Snippet: Southern hybridization analysis of RT-PCR products, showing the expression pattern of pg5 during infection of tomato plants by F. oxysporum f. sp. lycopersici . First-strand cDNAs were generated from total RNA isolated at the indicated time points (in hours) from roots and stems of uninfected or infected plants and used as templates for PCR with primers specific for pg5 (see Materials and Methods). Aliquots of the PCR products were electrophoresed on a 2% agarose gel, blotted onto a nylon membrane, and hybridized with the labeled pg5 probe. The position and size (in base pairs) of the pg5 fragment are indicated. The numbers represent days after inoculation. gDNA, genomic DNA.

Techniques Used: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Generated, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Labeling

56) Product Images from "Heparin at physiological concentration can enhance PEG-free in vitro infection with human hepatitis B virus"

Article Title: Heparin at physiological concentration can enhance PEG-free in vitro infection with human hepatitis B virus

Journal: Scientific Reports

doi: 10.1038/s41598-017-14573-9

Heparin facilitates PEG-free in vitro infection with HBV. ( a ) An illustration of experimental design. ( b ) Heparin alone can enhance HBV infection in a PEG-independent manner. The secretions of HBsAg and HBeAg were measured every other day by ELISA in a time course experiment (1–9 dpi). HepG2-NTCP-AS cells were infected with HBV serum pre-mixed with and without 4.5 μg/ml heparin. ( c ) A combinatory effect of heparin (4.5 µg/ml) and PEG (1.2%) can be detected by Southern blot analysis. Lane 1 and 2: Positive and negative controls were prepared from HBV DNA transfected HuH-7 cells and mock-infected HepG2-NTCP-AS cells, respectively. Heparin at a lower dose (4.5 μg/ml) enhanced HBV in vitro infection in the presence of 1.2% PEG (compare lanes 5 and 6), or in the absence of PEG (compare lanes 3 and 4). However, overdosed heparin (450 μg/ml) or treatment with PreS1 peptide (500 nM) was strongly inhibitory to HBV infection (lane 7 and 9). 4% PEG exhibited the most potent effect on HBV infection (lane 8). ( d ) In a PEG-free in vitro infection system, low dose heparin (4.5 µg/ml) significantly increased the percentage of HBc-positive HepG2-NTCP-AS cells by confocal microscopy. Cell nucleus was counterstained with DAPI. The estimated percentages of HBc positivity were based on the average from three different microscopic fields (Mean ± SD). Approximately 500 cells were scored in each field.
Figure Legend Snippet: Heparin facilitates PEG-free in vitro infection with HBV. ( a ) An illustration of experimental design. ( b ) Heparin alone can enhance HBV infection in a PEG-independent manner. The secretions of HBsAg and HBeAg were measured every other day by ELISA in a time course experiment (1–9 dpi). HepG2-NTCP-AS cells were infected with HBV serum pre-mixed with and without 4.5 μg/ml heparin. ( c ) A combinatory effect of heparin (4.5 µg/ml) and PEG (1.2%) can be detected by Southern blot analysis. Lane 1 and 2: Positive and negative controls were prepared from HBV DNA transfected HuH-7 cells and mock-infected HepG2-NTCP-AS cells, respectively. Heparin at a lower dose (4.5 μg/ml) enhanced HBV in vitro infection in the presence of 1.2% PEG (compare lanes 5 and 6), or in the absence of PEG (compare lanes 3 and 4). However, overdosed heparin (450 μg/ml) or treatment with PreS1 peptide (500 nM) was strongly inhibitory to HBV infection (lane 7 and 9). 4% PEG exhibited the most potent effect on HBV infection (lane 8). ( d ) In a PEG-free in vitro infection system, low dose heparin (4.5 µg/ml) significantly increased the percentage of HBc-positive HepG2-NTCP-AS cells by confocal microscopy. Cell nucleus was counterstained with DAPI. The estimated percentages of HBc positivity were based on the average from three different microscopic fields (Mean ± SD). Approximately 500 cells were scored in each field.

Techniques Used: In Vitro, Infection, Enzyme-linked Immunosorbent Assay, Southern Blot, Transfection, Confocal Microscopy

Establishment of an HBV in vitro infection system using a HepG2-NTCP-AS cell line. ( a ) An illustration of experimental design. ( b ) The expression of Flag-tagged NTCP in a HepG2-NTCP-AS stable cell line was detected by Western Blot using an anti-Flag antibody. De-glycosylation of NTCP by the PNGase F treatment shifted the MW from around 72 kD to 36 kD. The parental HepG2 cells served as a negative control for Flag-NTCP expression. GAPDH expression served as a loading control. ( c ) Similar trends of increasing expressions of both HBsAg and HBeAg were detected by ELISA only in HepG2-NTCP-AS cells infected with human HBV-containing serum. No detectable increase in HBsAg and HBeAg was noted in the negative controls using HepG2 cells or mock infection with no virus. ( d ) Total intracellular core particle-associated viral DNAs were analyzed by Southern blot at 9 dpi. Non-infected HepG2-NTCP-AS cells served as a negative control. RC: relaxed circle DNA; SS: single-strand DNA. ( e ) Confocal microscopy detected HBc protein (green) in HepG2-NTCP-AS cells infected with serum-derived HBV. Cell nuclei were counterstained with DAPI (blue).
Figure Legend Snippet: Establishment of an HBV in vitro infection system using a HepG2-NTCP-AS cell line. ( a ) An illustration of experimental design. ( b ) The expression of Flag-tagged NTCP in a HepG2-NTCP-AS stable cell line was detected by Western Blot using an anti-Flag antibody. De-glycosylation of NTCP by the PNGase F treatment shifted the MW from around 72 kD to 36 kD. The parental HepG2 cells served as a negative control for Flag-NTCP expression. GAPDH expression served as a loading control. ( c ) Similar trends of increasing expressions of both HBsAg and HBeAg were detected by ELISA only in HepG2-NTCP-AS cells infected with human HBV-containing serum. No detectable increase in HBsAg and HBeAg was noted in the negative controls using HepG2 cells or mock infection with no virus. ( d ) Total intracellular core particle-associated viral DNAs were analyzed by Southern blot at 9 dpi. Non-infected HepG2-NTCP-AS cells served as a negative control. RC: relaxed circle DNA; SS: single-strand DNA. ( e ) Confocal microscopy detected HBc protein (green) in HepG2-NTCP-AS cells infected with serum-derived HBV. Cell nuclei were counterstained with DAPI (blue).

Techniques Used: In Vitro, Infection, Expressing, Stable Transfection, Western Blot, Negative Control, Enzyme-linked Immunosorbent Assay, Southern Blot, Confocal Microscopy, Derivative Assay

57) Product Images from "HELQ promotes RAD51 paralog-dependent repair to avert germ cell attrition and tumourigenesis"

Article Title: HELQ promotes RAD51 paralog-dependent repair to avert germ cell attrition and tumourigenesis

Journal: Nature

doi: 10.1038/nature12565

HELQ interacts with DNA replication stress response factors a , Unique and total peptides identified by MS analysis of HELQ-FLAG co-IPs isolated from 293 cells. b , HELQ interaction network based on the MS results (coloured lines) and reported interactions from BIOGRID, STRING and MINT databases (dashed lines). c , Western blots of input and FLAG IPs from HELQ-FLAG and FLAG control samples. d , Reciprocal IP of endogenous RAD51C with HELQ-FLAG. e , Endogenous HELQ and RAD51C IPs from 293T cells. f , Purified, HIS-tagged BCDX2 complexes (top panel) were incubated with FLAG-complexed beads (bottom panels) to test for a direct interaction. FLAG and ALC1-FLAG are shown as negative controls. g , Western blot analysis of whole cells extracts (WCE) and chromatin fractions (Chr) from NIH3T3 HELQ-GFP expressing cells treated ± 100 ng/ml MMC for 24 hr.
Figure Legend Snippet: HELQ interacts with DNA replication stress response factors a , Unique and total peptides identified by MS analysis of HELQ-FLAG co-IPs isolated from 293 cells. b , HELQ interaction network based on the MS results (coloured lines) and reported interactions from BIOGRID, STRING and MINT databases (dashed lines). c , Western blots of input and FLAG IPs from HELQ-FLAG and FLAG control samples. d , Reciprocal IP of endogenous RAD51C with HELQ-FLAG. e , Endogenous HELQ and RAD51C IPs from 293T cells. f , Purified, HIS-tagged BCDX2 complexes (top panel) were incubated with FLAG-complexed beads (bottom panels) to test for a direct interaction. FLAG and ALC1-FLAG are shown as negative controls. g , Western blot analysis of whole cells extracts (WCE) and chromatin fractions (Chr) from NIH3T3 HELQ-GFP expressing cells treated ± 100 ng/ml MMC for 24 hr.

Techniques Used: Mass Spectrometry, Isolation, Western Blot, Purification, Incubation, Expressing

HELQ influences DNA repair and HR efficiency a , Lysates from immortalized mouse cells, grown under physiological O 2 and treated ± 3 μM APH for 6 hr, were probed for FANCD2. Wild-type (WT); HELQ-deficient (HQ); unmodified (S) and ubiquitinated (L) forms of FANCD2 . b , Chromatin fractions from immortalized mouse cells, probed for RAD51, histone H3 and α-tubulin at the indicated time points (hr) following treatment with ± 100 ng/ml MMC. c , Left panel, representative images of RAD51 focus formation in immortalized mouse cells at the indicated time points (hr) following treatment with 1 μM MMC. Right panel, quantification of RAD51 foci at the indicated time points. d , Pulsed field gel electrophoresis of genomic DNA from immortalized cells treated ± 1 μM MMC for 1 hr and recovered for the indicated number of hr. Undamaged (un). Wells: intact DNA; arrow: band containing large chromosomal fragments (10-0.45 Mb); below arrow: smaller fragments, resolved by size. e , HR frequencies in DR-GFP reporter cells treated with the indicated siRNAs. f , Clonogenic survival assays of immortalized mouse cells exposed to PARPi.
Figure Legend Snippet: HELQ influences DNA repair and HR efficiency a , Lysates from immortalized mouse cells, grown under physiological O 2 and treated ± 3 μM APH for 6 hr, were probed for FANCD2. Wild-type (WT); HELQ-deficient (HQ); unmodified (S) and ubiquitinated (L) forms of FANCD2 . b , Chromatin fractions from immortalized mouse cells, probed for RAD51, histone H3 and α-tubulin at the indicated time points (hr) following treatment with ± 100 ng/ml MMC. c , Left panel, representative images of RAD51 focus formation in immortalized mouse cells at the indicated time points (hr) following treatment with 1 μM MMC. Right panel, quantification of RAD51 foci at the indicated time points. d , Pulsed field gel electrophoresis of genomic DNA from immortalized cells treated ± 1 μM MMC for 1 hr and recovered for the indicated number of hr. Undamaged (un). Wells: intact DNA; arrow: band containing large chromosomal fragments (10-0.45 Mb); below arrow: smaller fragments, resolved by size. e , HR frequencies in DR-GFP reporter cells treated with the indicated siRNAs. f , Clonogenic survival assays of immortalized mouse cells exposed to PARPi.

Techniques Used: Pulsed-Field Gel, Electrophoresis

58) Product Images from "Sequence of the Escherichia coli O121 O-Antigen Gene Cluster and Detection of Enterohemorrhagic E. coli O121 by PCR Amplification of the wzx and wzy Genes"

Article Title: Sequence of the Escherichia coli O121 O-Antigen Gene Cluster and Detection of Enterohemorrhagic E. coli O121 by PCR Amplification of the wzx and wzy Genes

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.41.7.3379-3383.2003

Agarose gel showing PCR results of DNA from seven E. coli O121-positive swine fecal samples using two primer sets for the wzx gene and two primer sets for the wzy gene. (I) Lanes 1 to 7 and 8 to 14, PCR products using DNA from isolates K84-9 O121:H10, K84-11 O121:H10, K84-12 O121:H10, K84-36 O121:H10, K84-40 O121:H10, K102-27 O121:H − , and K150-1 O121:H − , respectively, and primer sets O121wzx1F-O121wzx1R (A) and O121wzx2F-O121wzx2R (B). Lanes 15 to 20 and 21 to 26, PCR products using DNA from E. coli O103:H3 93-0626, C. freundii ATCC 33128, C. braakii ATCC 43162, E. coli O111:NM 91.0130, S. flexneri ATCC 12022, and E. coli O121:H19 96-1585, respectively, and primer sets O121wzx2F-O121wzx2R and O121wzx1F-O121wzx1R, respectively. (II) Lanes 1 to 7 and 8 to 14, PCR products using DNA from the same swine fecal isolates described above for panel I and primer sets O121wzy1F-O121wzy1R (A) and O121wzy2F-O121wzy2R (B), respectively. Lanes 15 to 20 and 21 to 26, PCR products using DNA from E. coli O121:H19 96-1585, E. coli O103:H3 93-0626, C. freundii ATCC 33128, C. braakii ATCC 43162, E. coli O111:NM 91.0130, and S. flexneri ATCC 12022, respectively, and primer sets O121wzy1F-O121wzy1R and O121wzy2F-O121wzy2R, respectively. Lanes M, 100-bp ladder molecular size standards (Invitrogen).
Figure Legend Snippet: Agarose gel showing PCR results of DNA from seven E. coli O121-positive swine fecal samples using two primer sets for the wzx gene and two primer sets for the wzy gene. (I) Lanes 1 to 7 and 8 to 14, PCR products using DNA from isolates K84-9 O121:H10, K84-11 O121:H10, K84-12 O121:H10, K84-36 O121:H10, K84-40 O121:H10, K102-27 O121:H − , and K150-1 O121:H − , respectively, and primer sets O121wzx1F-O121wzx1R (A) and O121wzx2F-O121wzx2R (B). Lanes 15 to 20 and 21 to 26, PCR products using DNA from E. coli O103:H3 93-0626, C. freundii ATCC 33128, C. braakii ATCC 43162, E. coli O111:NM 91.0130, S. flexneri ATCC 12022, and E. coli O121:H19 96-1585, respectively, and primer sets O121wzx2F-O121wzx2R and O121wzx1F-O121wzx1R, respectively. (II) Lanes 1 to 7 and 8 to 14, PCR products using DNA from the same swine fecal isolates described above for panel I and primer sets O121wzy1F-O121wzy1R (A) and O121wzy2F-O121wzy2R (B), respectively. Lanes 15 to 20 and 21 to 26, PCR products using DNA from E. coli O121:H19 96-1585, E. coli O103:H3 93-0626, C. freundii ATCC 33128, C. braakii ATCC 43162, E. coli O111:NM 91.0130, and S. flexneri ATCC 12022, respectively, and primer sets O121wzy1F-O121wzy1R and O121wzy2F-O121wzy2R, respectively. Lanes M, 100-bp ladder molecular size standards (Invitrogen).

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction

59) Product Images from "Integron mediated multidrug resistance in extended spectrum beta-lactamase producing clinical isolates of Klebsiella pneumoniae"

Article Title: Integron mediated multidrug resistance in extended spectrum beta-lactamase producing clinical isolates of Klebsiella pneumoniae

Journal: Brazilian Journal of Microbiology

doi:

PCR amplification of class 1 integron variable regions in ESBL producing Klebsiella pneumoniae isolates (Lanes 1–5 and 7–12). Lane 6, DNA molecular weight marker (100 bp, Fermentas).
Figure Legend Snippet: PCR amplification of class 1 integron variable regions in ESBL producing Klebsiella pneumoniae isolates (Lanes 1–5 and 7–12). Lane 6, DNA molecular weight marker (100 bp, Fermentas).

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

PCR amplification product of class 1 (A) and class 2 (B) integrase genes in a number of ESBL producing Klebsiella pneumoniae clinical isolates. Ladder, DNA molecular weight marker (100 bp, Fermentas); C+, positive control for class 1 integron; C − , negative control (no DNA template).
Figure Legend Snippet: PCR amplification product of class 1 (A) and class 2 (B) integrase genes in a number of ESBL producing Klebsiella pneumoniae clinical isolates. Ladder, DNA molecular weight marker (100 bp, Fermentas); C+, positive control for class 1 integron; C − , negative control (no DNA template).

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

60) Product Images from "Molecular Diagnosis of Disseminated Adiaspiromycosis Due to Emmonsia crescens ▿"

Article Title: Molecular Diagnosis of Disseminated Adiaspiromycosis Due to Emmonsia crescens ▿

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.01885-08

Amplification patterns of DNA extracted from pulmonary samples of a patient suffering from adiaspiromycosis. Lane 1, 100-bp ladder; lane 2, negative control (water); lane 3, BAL sample; lane 4, tracheal aspiration; lane 5, transbronchial biopsy; lane
Figure Legend Snippet: Amplification patterns of DNA extracted from pulmonary samples of a patient suffering from adiaspiromycosis. Lane 1, 100-bp ladder; lane 2, negative control (water); lane 3, BAL sample; lane 4, tracheal aspiration; lane 5, transbronchial biopsy; lane

Techniques Used: Amplification, Negative Control

61) Product Images from "Genomic Characterization of Interspecific Hybrids between the Scallops Argopecten purpuratus and A. irradians irradians"

Article Title: Genomic Characterization of Interspecific Hybrids between the Scallops Argopecten purpuratus and A. irradians irradians

Journal: PLoS ONE

doi: 10.1371/journal.pone.0062432

Examples of allotriploid (a) and aneuploid (b) in the F1 hybrids examined by GISH. (a) Chromosomes are identified using the A. i. irradians genomic DNA probes. The 16 (n) chromosomes originating from A. i. irradians are shown with rhodamine (red), and the other 32 (2 n) chromosomes originating from A. purpuratus are counterstained with DAPI (blue). (b) Chromosomes are identified using the A. purpuratus genomic DNA probes. The green chromosomes are from A. purpuratus , and the red ones are from A. i. irradians . One chromosome from A. purpuratus and one chromosome from A. i. irradians were eliminated in the metaphase spread. Bars = 5 µm.
Figure Legend Snippet: Examples of allotriploid (a) and aneuploid (b) in the F1 hybrids examined by GISH. (a) Chromosomes are identified using the A. i. irradians genomic DNA probes. The 16 (n) chromosomes originating from A. i. irradians are shown with rhodamine (red), and the other 32 (2 n) chromosomes originating from A. purpuratus are counterstained with DAPI (blue). (b) Chromosomes are identified using the A. purpuratus genomic DNA probes. The green chromosomes are from A. purpuratus , and the red ones are from A. i. irradians . One chromosome from A. purpuratus and one chromosome from A. i. irradians were eliminated in the metaphase spread. Bars = 5 µm.

Techniques Used:

Representative metaphase chromosomes of the F1 hybrids of A. purpuratus × A. i. irradians examined by GISH. In (a), the total genomic DNA from A. i. irradians is used as the probe. The chromosomes are stained with rhodamine (red) and counterstained with DAPI (blue). In (b), the total genomic DNA from A. purpuratus is used as the probe. The chromosomes are stained with FITC (green) and counterstained with PI (red). Bars = 5 µm.
Figure Legend Snippet: Representative metaphase chromosomes of the F1 hybrids of A. purpuratus × A. i. irradians examined by GISH. In (a), the total genomic DNA from A. i. irradians is used as the probe. The chromosomes are stained with rhodamine (red) and counterstained with DAPI (blue). In (b), the total genomic DNA from A. purpuratus is used as the probe. The chromosomes are stained with FITC (green) and counterstained with PI (red). Bars = 5 µm.

Techniques Used: Staining

62) Product Images from "Mice with a Targeted Disruption of the Cl−/HCO3− Exchanger AE3 Display a Reduced Seizure Threshold"

Article Title: Mice with a Targeted Disruption of the Cl−/HCO3− Exchanger AE3 Display a Reduced Seizure Threshold

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.26.1.182-191.2006

Targeted disruption of the murine Slc4a3 gene. (A) Schematic diagram of the murine Slc4a3 locus. By homologous recombination of the targeting construct, a neomycin resistance cassette flanked by two loxP sites and a third loxP site with an additional EcoRV restriction site was introduced into the Slc4a3 locus. After transient expression of cre recombinase, clones with a deletion including exons 6 to 19 were used for the generation of AE3-deficient mice. (B) Genotyping of tail biopsy DNA from wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice by PCR. (C) Southern blot analysis with a probe located at the 3′ end of Slc4a3 (A) resulted in a ∼4.0-kb band, compared to ∼18.0 kb for the wild-type allele. (D) Northern blot analysis of total RNA from 3-month-old animals with a full-length AE3-cDNA as a probe demonstrated complete absence of AE3 transcripts in knockout brain and heart. (E) Western blot analysis of brain and heart membrane-enriched fractions from 3-month-old animals confirmed this result at the protein level. Note the differing molecular masses of the brain and the heart variant as indicated by the arrows. The bands of lower molecular size represent unspecific cross-reactivity of the antibody.
Figure Legend Snippet: Targeted disruption of the murine Slc4a3 gene. (A) Schematic diagram of the murine Slc4a3 locus. By homologous recombination of the targeting construct, a neomycin resistance cassette flanked by two loxP sites and a third loxP site with an additional EcoRV restriction site was introduced into the Slc4a3 locus. After transient expression of cre recombinase, clones with a deletion including exons 6 to 19 were used for the generation of AE3-deficient mice. (B) Genotyping of tail biopsy DNA from wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice by PCR. (C) Southern blot analysis with a probe located at the 3′ end of Slc4a3 (A) resulted in a ∼4.0-kb band, compared to ∼18.0 kb for the wild-type allele. (D) Northern blot analysis of total RNA from 3-month-old animals with a full-length AE3-cDNA as a probe demonstrated complete absence of AE3 transcripts in knockout brain and heart. (E) Western blot analysis of brain and heart membrane-enriched fractions from 3-month-old animals confirmed this result at the protein level. Note the differing molecular masses of the brain and the heart variant as indicated by the arrows. The bands of lower molecular size represent unspecific cross-reactivity of the antibody.

Techniques Used: Homologous Recombination, Construct, Expressing, Clone Assay, Mouse Assay, Knock-Out, Polymerase Chain Reaction, Southern Blot, Northern Blot, Western Blot, Variant Assay

63) Product Images from "Characteristics of Age-Related Changes in Cultured Human Vocal Fold Fibroblasts"

Article Title: Characteristics of Age-Related Changes in Cultured Human Vocal Fold Fibroblasts

Journal:

doi: 10.1097/MLG.0b013e31817aec6c

Terminal restriction fragment (telomere) length in cultures human vocal fold fibroblasts (21T, 59T and 79T) during passage 7 to 18 by Southern blot analysis. Genomic DNA was isolated from hVFF of passage 7, 13 and 18, and hybridized with a telomeric probe
Figure Legend Snippet: Terminal restriction fragment (telomere) length in cultures human vocal fold fibroblasts (21T, 59T and 79T) during passage 7 to 18 by Southern blot analysis. Genomic DNA was isolated from hVFF of passage 7, 13 and 18, and hybridized with a telomeric probe

Techniques Used: Southern Blot, Isolation

Related Articles

Amplification:

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Article Title: Caspase-2-mediated cell death is required for deleting aneuploid cells
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Article Title: Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter
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Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
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Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization
Article Snippet: Genomic DNA was subjected to quantitative real-time PCR using a Lightcycler480 and SYBR Green system (Roche Diagnostic Systems) following the manufacturer’s protocol. .. Amplification of vector plasmid DNA for generation of standard curves was performed using concentrations of plasmids ranging from 107 molecules/µL to 10 molecules/µL as determined by spectrophotometry.

High Throughput Screening Assay:

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
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Synthesized:

Article Title: NKL homeobox gene NKX2-2 is aberrantly expressed in Hodgkin lymphoma
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Cytometry:

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
Article Snippet: IDLV Integration Site Analysis GFP expression of transduced cells was determined by flow cytometry after transfection with nucleases. .. Genomic DNA was isolated using the High Pure PCR Template Preparation Kit (Roche) after 3–4 weeks of cultivation when the frequency of GFP-expressing cells was stable.

Blocking Assay:

Article Title: ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development
Article Snippet: .. Genomic DNA from WT-F and ZIPCO-ko were digested with Pci I and Kpn I. Membrane pre-hybridization, blocking, hybridization and detection were carried out according to manufacturer's instructions (Roche). .. Primers used in this study Primers used for cloning, generating probes for Southern analysis, sequencing, RT-PCR of zipco transcripts and qPCR analysis are indicated in Supplementary Table S3.

Article Title: Characterization of morphology and resistance to Blumeria graminis of winter triticale monosomic addition lines with chromosome 2D of Aegilopstauschii
Article Snippet: Genomic DNA from Ae. tauschii and T . monococcum were labeled by nick translation (using NickTranslation Kit, Roche, Mannheim, Germany) with tetramethyl-5-dUTP-rhodamine (Roche) or digoxigenin-11-dUTP (Roche) depending on the visualization concept. .. Blocking DNA from triticale, Ae . speltoides and S . cereale , was sheared to fragments of 5–10 kb by boiling for 30–45 min and used at a ratio of 1:50 (probe:block).

Real-time Polymerase Chain Reaction:

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization
Article Snippet: .. Genomic DNA was subjected to quantitative real-time PCR using a Lightcycler480 and SYBR Green system (Roche Diagnostic Systems) following the manufacturer’s protocol. .. Amplification of vector plasmid DNA for generation of standard curves was performed using concentrations of plasmids ranging from 107 molecules/µL to 10 molecules/µL as determined by spectrophotometry.

Incubation:

Article Title: Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides 1 1 [W] 1 [W] [OPEN]
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Luciferase:

Article Title: Transcriptional Regulation of PIK3CA Oncogene by NF-?B in Ovarian Cancer Microenvironment
Article Snippet: .. To construct luciferase reporter plasmids, 5′TRRs of the human and mouse PIK3CA were amplified from BAC or genomic DNA using Expand High Fidelity PCR System (Roche, Indianapolis, IN) and inserted upstream of the luciferase gene in pGL3-Basic vector (Promega, Madison, WI). .. Sequences of 5′TRR in all these reporter constructs were verified by DNA sequencing.

In Silico:

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
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Expressing:

Article Title: Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter
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Article Title: NKL homeobox gene NKX2-2 is aberrantly expressed in Hodgkin lymphoma
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Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
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Article Title: Transcriptional Regulation of PIK3CA Oncogene by NF-?B in Ovarian Cancer Microenvironment
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Transformation Assay:

Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
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Hybridization:

Article Title: ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development
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Flow Cytometry:

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
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Southern Blot:

Article Title: ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development
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Article Title: Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides 1 1 [W] 1 [W] [OPEN]
Article Snippet: Paragraph title: Southern-Blot Analysis ... For each sample, 15 µg genomic DNA was digested with Bam HI or Cla I restriction endonucleases ( ) and electrophoresed in 0.8% (w/v) agarose gels for 22 h. All gels were loaded with 3 to 4 µL of digoxigenin (DIG)-labeled λ DNA marker III ( Hin dIII- Eco RI digested, Roche Applied Science) as size marker and 100 picogram mL–1 Cla I- or Bam HI-digested R4pGWB501 plasmid DNA containing P. vittata actin promoter and GUSPlus, both of which can be detected in x-ray film along with experimental samples.

Nick Translation:

Article Title: Characterization of morphology and resistance to Blumeria graminis of winter triticale monosomic addition lines with chromosome 2D of Aegilopstauschii
Article Snippet: .. Genomic DNA from Ae. tauschii and T . monococcum were labeled by nick translation (using NickTranslation Kit, Roche, Mannheim, Germany) with tetramethyl-5-dUTP-rhodamine (Roche) or digoxigenin-11-dUTP (Roche) depending on the visualization concept. .. Blocking DNA from triticale, Ae . speltoides and S . cereale , was sheared to fragments of 5–10 kb by boiling for 30–45 min and used at a ratio of 1:50 (probe:block).

Cell Culture:

Article Title: CpG island methylation is a common finding in colorectal cancer cell lines
Article Snippet: Cells were cultured in DMEM, EMEM or RPMI, all supplemented with 10% foetal calf serum, and were harvested when growing exponentially. .. Genomic DNA (500 ng) was used in PCR with 0.5 μ M of each of the primers 5′-ATATAAACTTGTGGTAGTTCCAGCTGGT-3′ and 5′-ATCAAAGAATGGTCCTGCACC-3′, 2.5 mM MgCl2 , 100 μ M dNTPs, 1.25 U FastStart TaqDNA polymerase (Roche Diagnostics GmBH, Mannheim, Germany) in the reaction buffer provided by the manufacturer.

Overlap Extension Polymerase Chain Reaction:

Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
Article Snippet: Paragraph title: Splicing by Overlap Extension (SOE) PCR ... The upstream and downstream fragments of orfU gene were amplified from gDNA of E7946 strain; gDNA of E7946 was extracted and purified using High Pure PCR Template Preparation Kit (Roche Life Science, Indianapolis, IN, United States).

DNA Sequencing:

Article Title: Transcriptional Regulation of PIK3CA Oncogene by NF-?B in Ovarian Cancer Microenvironment
Article Snippet: To construct luciferase reporter plasmids, 5′TRRs of the human and mouse PIK3CA were amplified from BAC or genomic DNA using Expand High Fidelity PCR System (Roche, Indianapolis, IN) and inserted upstream of the luciferase gene in pGL3-Basic vector (Promega, Madison, WI). .. Sequences of 5′TRR in all these reporter constructs were verified by DNA sequencing.

Sequencing:

Article Title: Glycoprotein Ibα Kozak polymorphism in patients presenting with early-onset acute coronary syndrome
Article Snippet: Blood samples were obtained and genomic DNA was isolated with the High Pure PCR Template Preparation kit (Roche Diagnostics, IN, USA). .. Genotyping for GPIbα Kozak was performed by PCR based on the GPIbα gene sequence.

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
Article Snippet: Genomic DNA was isolated using the High Pure PCR Template Preparation Kit (Roche) after 3–4 weeks of cultivation when the frequency of GFP-expressing cells was stable. .. For sequencing with the Illumina MiSeq platform, barcode-containing adapters were added to LAM-PCR products by an additional PCR step, and the integration site data were analyzed using the high-throughput insertion site analysis pipeline (HISAP).

Binding Assay:

Article Title: NKL homeobox gene NKX2-2 is aberrantly expressed in Hodgkin lymphoma
Article Snippet: For normalization of expression levels we analyzed the transcript of TATA box binding protein (TBP). .. For genomic copy number quantification of exon 1 from SMURF2 we extracted genomic DNA using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany).

BAC Assay:

Article Title: Transcriptional Regulation of PIK3CA Oncogene by NF-?B in Ovarian Cancer Microenvironment
Article Snippet: .. To construct luciferase reporter plasmids, 5′TRRs of the human and mouse PIK3CA were amplified from BAC or genomic DNA using Expand High Fidelity PCR System (Roche, Indianapolis, IN) and inserted upstream of the luciferase gene in pGL3-Basic vector (Promega, Madison, WI). .. Sequences of 5′TRR in all these reporter constructs were verified by DNA sequencing.

Methylation:

Article Title: DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO
Article Snippet: Methylated DNA was purified and amplified from C. elegans gDNA much as previously described ( ). .. Dpn I was inactivated (80°C, 20 min) and genomic DNA ligated to double-stranded adaptors with T4-ligase (Roche).

Mutagenesis:

Article Title: CpG island methylation is a common finding in colorectal cancer cell lines
Article Snippet: Genomic DNA (500 ng) was used in PCR with 0.5 μ M of each of the primers 5′-ATATAAACTTGTGGTAGTTCCAGCTGGT-3′ and 5′-ATCAAAGAATGGTCCTGCACC-3′, 2.5 mM MgCl2 , 100 μ M dNTPs, 1.25 U FastStart TaqDNA polymerase (Roche Diagnostics GmBH, Mannheim, Germany) in the reaction buffer provided by the manufacturer. .. The resistance of PCR amplicons to Bsl I digestion indicated the presence of a mutation at the first or second base of Ki-ras codon 13.

Article Title: Caspase-2-mediated cell death is required for deleting aneuploid cells
Article Snippet: .. For genotyping of mutant mice including founder mice, genomic DNA was extracted from postnatal day 10 tail tissue using High Pure PCR Template Preparation Kit (ROCHE, Basel Schweiz, Switzerland). .. A 497 bp fragment containing the putative mutation was amplified by PCR and digested with XhoI restriction enzyme.

Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
Article Snippet: For example, to create a null mutation in the orfU gene (1284-bp) in CTX prophage in the background of V. cholerae O1 strain E7946 , a two-step SOE PCR amplification protocol was engineered. .. The upstream and downstream fragments of orfU gene were amplified from gDNA of E7946 strain; gDNA of E7946 was extracted and purified using High Pure PCR Template Preparation Kit (Roche Life Science, Indianapolis, IN, United States).

Article Title: Development of a Highly Sensitive and Specific Method for Detection of Circulating Tumor Cells Harboring Somatic Mutations in Non-Small-Cell Lung Cancer Patients
Article Snippet: .. Genomic DNA from samples displaying a strong EGFR DelEx19 mutation signal in our newly developed assay (shown in ), was analyzed on a Roche 454 GS FLX+ Titanium sequencer. ..

Isolation:

Article Title: Glycoprotein Ibα Kozak polymorphism in patients presenting with early-onset acute coronary syndrome
Article Snippet: .. Blood samples were obtained and genomic DNA was isolated with the High Pure PCR Template Preparation kit (Roche Diagnostics, IN, USA). .. The quantity and quality of DNA in each sample were estimated by UV absorbance and agarose gel electrophoresis.

Article Title: DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO
Article Snippet: Briefly, genomic DNA was isolated using a DNAeasy kit (Qiagen) and then digested overnight with Dpn I (New England Biolabs (NEB)) to cut at GAm TC sites. .. Dpn I was inactivated (80°C, 20 min) and genomic DNA ligated to double-stranded adaptors with T4-ligase (Roche).

Article Title: NKL homeobox gene NKX2-2 is aberrantly expressed in Hodgkin lymphoma
Article Snippet: Primary human total RNA used in this study was commercially obtained - isolated from peripheral blood mononuclear cells (PBC), thymus, lymph node (LN), spleen, bone marrow (BM), NK-cells, and brain from Biochain/BioCat (Heidelberg, Germany), and RNA from peripheral CD19-positive B-cells, CD3-positive T-cells, and CD34-positive stem cells from Miltenyi Biotec (Bergisch Gladbach, Germany). cDNA was synthesized from 5 μg RNA by random priming using Superscript II (Invitrogen). .. For genomic copy number quantification of exon 1 from SMURF2 we extracted genomic DNA using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany).

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
Article Snippet: .. Genomic DNA was isolated using the High Pure PCR Template Preparation Kit (Roche) after 3–4 weeks of cultivation when the frequency of GFP-expressing cells was stable. .. Each sample was analyzed by at least two repetitions of 3′ LAM-PCR with enzymes MseI and MluCI and 3′ nrLAM-PCR using 500 ng of genomic DNA each.

Article Title: Characterization of a Novel Type II Restriction-Modification System, Sth368I, Encoded by the Integrative Element ICESt1 of Streptococcus thermophilus CNRZ368
Article Snippet: Paragraph title: Isolation, partial purification, and test of endonuclease extracts. ... The reaction mixture (20 μl) contained 0.2 μg of pBC KS(+) DNA or 0.5 μg of recombinant λ or genomic DNA, 10 μl of cell extract, and reaction buffer B (Roche Diagnostics).

Transfection:

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
Article Snippet: IDLV Integration Site Analysis GFP expression of transduced cells was determined by flow cytometry after transfection with nucleases. .. Genomic DNA was isolated using the High Pure PCR Template Preparation Kit (Roche) after 3–4 weeks of cultivation when the frequency of GFP-expressing cells was stable.

Labeling:

Article Title: Characterization of morphology and resistance to Blumeria graminis of winter triticale monosomic addition lines with chromosome 2D of Aegilopstauschii
Article Snippet: .. Genomic DNA from Ae. tauschii and T . monococcum were labeled by nick translation (using NickTranslation Kit, Roche, Mannheim, Germany) with tetramethyl-5-dUTP-rhodamine (Roche) or digoxigenin-11-dUTP (Roche) depending on the visualization concept. .. Blocking DNA from triticale, Ae . speltoides and S . cereale , was sheared to fragments of 5–10 kb by boiling for 30–45 min and used at a ratio of 1:50 (probe:block).

Purification:

Article Title: DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO
Article Snippet: Methylated DNA was purified and amplified from C. elegans gDNA much as previously described ( ). .. Dpn I was inactivated (80°C, 20 min) and genomic DNA ligated to double-stranded adaptors with T4-ligase (Roche).

Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
Article Snippet: .. The upstream and downstream fragments of orfU gene were amplified from gDNA of E7946 strain; gDNA of E7946 was extracted and purified using High Pure PCR Template Preparation Kit (Roche Life Science, Indianapolis, IN, United States). .. All the PCR primers are listed in .

Article Title: Characterization of a Novel Type II Restriction-Modification System, Sth368I, Encoded by the Integrative Element ICESt1 of Streptococcus thermophilus CNRZ368
Article Snippet: Paragraph title: Isolation, partial purification, and test of endonuclease extracts. ... The reaction mixture (20 μl) contained 0.2 μg of pBC KS(+) DNA or 0.5 μg of recombinant λ or genomic DNA, 10 μl of cell extract, and reaction buffer B (Roche Diagnostics).

Polymerase Chain Reaction:

Article Title: Glycoprotein Ibα Kozak polymorphism in patients presenting with early-onset acute coronary syndrome
Article Snippet: .. Blood samples were obtained and genomic DNA was isolated with the High Pure PCR Template Preparation kit (Roche Diagnostics, IN, USA). .. The quantity and quality of DNA in each sample were estimated by UV absorbance and agarose gel electrophoresis.

Article Title: DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO
Article Snippet: Dpn I was inactivated (80°C, 20 min) and genomic DNA ligated to double-stranded adaptors with T4-ligase (Roche). .. DNA fragments were then digested with Dpn II (NEB) for 1 h at 37°C to cut unmethylated GATC sites thereby preventing PCR amplification of unmethylated DNA.

Article Title: CpG island methylation is a common finding in colorectal cancer cell lines
Article Snippet: .. Genomic DNA (500 ng) was used in PCR with 0.5 μ M of each of the primers 5′-ATATAAACTTGTGGTAGTTCCAGCTGGT-3′ and 5′-ATCAAAGAATGGTCCTGCACC-3′, 2.5 mM MgCl2 , 100 μ M dNTPs, 1.25 U FastStart TaqDNA polymerase (Roche Diagnostics GmBH, Mannheim, Germany) in the reaction buffer provided by the manufacturer. .. PCR cycling conditions were as follows: 95°C for 5 min; 10 cycles of 95°C for 20 s, 70°C for 20 s with a touchdown of 1°C per cycle; followed by a further 32 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 30 s prior to a final extension at 72°C for 4 min.

Article Title: Caspase-2-mediated cell death is required for deleting aneuploid cells
Article Snippet: .. For genotyping of mutant mice including founder mice, genomic DNA was extracted from postnatal day 10 tail tissue using High Pure PCR Template Preparation Kit (ROCHE, Basel Schweiz, Switzerland). .. A 497 bp fragment containing the putative mutation was amplified by PCR and digested with XhoI restriction enzyme.

Article Title: Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter
Article Snippet: .. To assure that the differences in expression did not result from disparate efficiencies of the hUAT versus hUAT2 primer pairs, PCR was performed with these primers using genomic DNA as a template with the Expand Long Template PCR kit in buffer number 1 (Roche Molecular Biochemicals). .. Since expression of hUAT was much greater than hUAT2 in the MTC panels, tissue levels of hUAT expression were determined by Northern analysis and dot-blot array.

Article Title: NKL homeobox gene NKX2-2 is aberrantly expressed in Hodgkin lymphoma
Article Snippet: .. For genomic copy number quantification of exon 1 from SMURF2 we extracted genomic DNA using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany). ..

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
Article Snippet: .. Genomic DNA was isolated using the High Pure PCR Template Preparation Kit (Roche) after 3–4 weeks of cultivation when the frequency of GFP-expressing cells was stable. .. Each sample was analyzed by at least two repetitions of 3′ LAM-PCR with enzymes MseI and MluCI and 3′ nrLAM-PCR using 500 ng of genomic DNA each.

Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
Article Snippet: .. The upstream and downstream fragments of orfU gene were amplified from gDNA of E7946 strain; gDNA of E7946 was extracted and purified using High Pure PCR Template Preparation Kit (Roche Life Science, Indianapolis, IN, United States). .. All the PCR primers are listed in .

Article Title: ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development
Article Snippet: Genomic analysis of recombinant parasites Southern blot analysis was performed on WT, WT-F, ZIPCO, ZIPCO-F, ZIPCO-ko and ZIPCO-HA recombinant parasites as previously described (Lacroix et al , ) using a Digoxygenin-labelled PCR-Probe and hybridization kit (Roche). .. Genomic DNA from WT-F and ZIPCO-ko were digested with Pci I and Kpn I. Membrane pre-hybridization, blocking, hybridization and detection were carried out according to manufacturer's instructions (Roche).

Article Title: Transcriptional Regulation of PIK3CA Oncogene by NF-?B in Ovarian Cancer Microenvironment
Article Snippet: .. To construct luciferase reporter plasmids, 5′TRRs of the human and mouse PIK3CA were amplified from BAC or genomic DNA using Expand High Fidelity PCR System (Roche, Indianapolis, IN) and inserted upstream of the luciferase gene in pGL3-Basic vector (Promega, Madison, WI). .. Sequences of 5′TRR in all these reporter constructs were verified by DNA sequencing.

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization
Article Snippet: Genomic DNA was subjected to quantitative real-time PCR using a Lightcycler480 and SYBR Green system (Roche Diagnostic Systems) following the manufacturer’s protocol. .. Plasmid DNA used for standard curve generation was diluted with genomic DNA from uninfected 293T cells to control for any inhibitory effect of genomic DNA on PCR.

Construct:

Article Title: Transcriptional Regulation of PIK3CA Oncogene by NF-?B in Ovarian Cancer Microenvironment
Article Snippet: .. To construct luciferase reporter plasmids, 5′TRRs of the human and mouse PIK3CA were amplified from BAC or genomic DNA using Expand High Fidelity PCR System (Roche, Indianapolis, IN) and inserted upstream of the luciferase gene in pGL3-Basic vector (Promega, Madison, WI). .. Sequences of 5′TRR in all these reporter constructs were verified by DNA sequencing.

Mouse Assay:

Article Title: Caspase-2-mediated cell death is required for deleting aneuploid cells
Article Snippet: .. For genotyping of mutant mice including founder mice, genomic DNA was extracted from postnatal day 10 tail tissue using High Pure PCR Template Preparation Kit (ROCHE, Basel Schweiz, Switzerland). .. A 497 bp fragment containing the putative mutation was amplified by PCR and digested with XhoI restriction enzyme.

Plasmid Preparation:

Article Title: DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO
Article Snippet: DamID analysis In the main experiment described here, the two DamID strains were subjected to daf-2 RNAi from hatching at 20°C, much as previously described , and using the same daf-2 RNAi feeding plasmid. .. Dpn I was inactivated (80°C, 20 min) and genomic DNA ligated to double-stranded adaptors with T4-ligase (Roche).

Article Title: Caspase-2-mediated cell death is required for deleting aneuploid cells
Article Snippet: To produce Cas9 mRNA, Cas9 plasmid was linearized using XhoI restriction enzyme (NEB) and mRNA was transcribed using mMessage mMachine T7 Ultra Kit (Life technologies, Carlsbad, CA, USA) followed by capping and polyadenylation. .. For genotyping of mutant mice including founder mice, genomic DNA was extracted from postnatal day 10 tail tissue using High Pure PCR Template Preparation Kit (ROCHE, Basel Schweiz, Switzerland).

Article Title: Transcriptional Regulation of PIK3CA Oncogene by NF-?B in Ovarian Cancer Microenvironment
Article Snippet: .. To construct luciferase reporter plasmids, 5′TRRs of the human and mouse PIK3CA were amplified from BAC or genomic DNA using Expand High Fidelity PCR System (Roche, Indianapolis, IN) and inserted upstream of the luciferase gene in pGL3-Basic vector (Promega, Madison, WI). .. Sequences of 5′TRR in all these reporter constructs were verified by DNA sequencing.

Article Title: Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides 1 1 [W] 1 [W] [OPEN]
Article Snippet: .. For each sample, 15 µg genomic DNA was digested with Bam HI or Cla I restriction endonucleases ( ) and electrophoresed in 0.8% (w/v) agarose gels for 22 h. All gels were loaded with 3 to 4 µL of digoxigenin (DIG)-labeled λ DNA marker III ( Hin dIII- Eco RI digested, Roche Applied Science) as size marker and 100 picogram mL–1 Cla I- or Bam HI-digested R4pGWB501 plasmid DNA containing P. vittata actin promoter and GUSPlus, both of which can be detected in x-ray film along with experimental samples. .. DNA was transferred to a nylon membrane (GE Healthcare) by capillary blotting ( ) and cross linked by UV irradiation.

SYBR Green Assay:

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization
Article Snippet: .. Genomic DNA was subjected to quantitative real-time PCR using a Lightcycler480 and SYBR Green system (Roche Diagnostic Systems) following the manufacturer’s protocol. .. Amplification of vector plasmid DNA for generation of standard curves was performed using concentrations of plasmids ranging from 107 molecules/µL to 10 molecules/µL as determined by spectrophotometry.

Multiplex Assay:

Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
Article Snippet: Splicing by Overlap Extension (SOE) PCR We used splicing by overlap extension (SOE) PCR and multiplex genome editing by natural transformation to create our desired mutation(s) in V. cholerae strains as described previously ( ; ). .. The upstream and downstream fragments of orfU gene were amplified from gDNA of E7946 strain; gDNA of E7946 was extracted and purified using High Pure PCR Template Preparation Kit (Roche Life Science, Indianapolis, IN, United States).

Recombinant:

Article Title: ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development
Article Snippet: Paragraph title: Genomic analysis of recombinant parasites ... Genomic DNA from WT-F and ZIPCO-ko were digested with Pci I and Kpn I. Membrane pre-hybridization, blocking, hybridization and detection were carried out according to manufacturer's instructions (Roche).

Article Title: Characterization of a Novel Type II Restriction-Modification System, Sth368I, Encoded by the Integrative Element ICESt1 of Streptococcus thermophilus CNRZ368
Article Snippet: .. The reaction mixture (20 μl) contained 0.2 μg of pBC KS(+) DNA or 0.5 μg of recombinant λ or genomic DNA, 10 μl of cell extract, and reaction buffer B (Roche Diagnostics). ..

Agarose Gel Electrophoresis:

Article Title: Glycoprotein Ibα Kozak polymorphism in patients presenting with early-onset acute coronary syndrome
Article Snippet: Blood samples were obtained and genomic DNA was isolated with the High Pure PCR Template Preparation kit (Roche Diagnostics, IN, USA). .. The quantity and quality of DNA in each sample were estimated by UV absorbance and agarose gel electrophoresis.

Size-exclusion Chromatography:

Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
Article Snippet: The upstream and downstream fragments of orfU gene were amplified from gDNA of E7946 strain; gDNA of E7946 was extracted and purified using High Pure PCR Template Preparation Kit (Roche Life Science, Indianapolis, IN, United States). .. Q5 High-Fidelity DNA Polymerase (New England Biolabs Inc., Ipswich, MA, United States) was used to amplify the desired DNA fragments and the PCR conditions were as follows: initial denaturation at 98°C for 3 min, 30 cycles each of denaturation at 98°C for 30 sec, annealing at 60°C for 45 sec and elongation at 72°C for 2 min, and final extension at 72°C for 5 min followed by hold at 4°C.

Laser Capture Microdissection:

Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
Article Snippet: Genomic DNA was isolated using the High Pure PCR Template Preparation Kit (Roche) after 3–4 weeks of cultivation when the frequency of GFP-expressing cells was stable. .. Each sample was analyzed by at least two repetitions of 3′ LAM-PCR with enzymes MseI and MluCI and 3′ nrLAM-PCR using 500 ng of genomic DNA each.

Spectrophotometry:

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization
Article Snippet: Genomic DNA was subjected to quantitative real-time PCR using a Lightcycler480 and SYBR Green system (Roche Diagnostic Systems) following the manufacturer’s protocol. .. Amplification of vector plasmid DNA for generation of standard curves was performed using concentrations of plasmids ranging from 107 molecules/µL to 10 molecules/µL as determined by spectrophotometry.

Irradiation:

Article Title: Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides 1 1 [W] 1 [W] [OPEN]
Article Snippet: For each sample, 15 µg genomic DNA was digested with Bam HI or Cla I restriction endonucleases ( ) and electrophoresed in 0.8% (w/v) agarose gels for 22 h. All gels were loaded with 3 to 4 µL of digoxigenin (DIG)-labeled λ DNA marker III ( Hin dIII- Eco RI digested, Roche Applied Science) as size marker and 100 picogram mL–1 Cla I- or Bam HI-digested R4pGWB501 plasmid DNA containing P. vittata actin promoter and GUSPlus, both of which can be detected in x-ray film along with experimental samples. .. DNA was transferred to a nylon membrane (GE Healthcare) by capillary blotting ( ) and cross linked by UV irradiation.

DNA Purification:

Article Title: Characterization of morphology and resistance to Blumeria graminis of winter triticale monosomic addition lines with chromosome 2D of Aegilopstauschii
Article Snippet: Probe labeling Total genomic DNA was extracted from fresh leaves of Ae . tauschii (DD), Triticum monococcum (Am Am ), Ae . speltoides (BB), S . cereale (RR), and triticale ‘Bogo’ (AABBRR) using GeneMATRIX Plant and Funghi DNA Purification Kit (EURx Ltd.). .. Genomic DNA from Ae. tauschii and T . monococcum were labeled by nick translation (using NickTranslation Kit, Roche, Mannheim, Germany) with tetramethyl-5-dUTP-rhodamine (Roche) or digoxigenin-11-dUTP (Roche) depending on the visualization concept.

Marker:

Article Title: Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides 1 1 [W] 1 [W] [OPEN]
Article Snippet: .. For each sample, 15 µg genomic DNA was digested with Bam HI or Cla I restriction endonucleases ( ) and electrophoresed in 0.8% (w/v) agarose gels for 22 h. All gels were loaded with 3 to 4 µL of digoxigenin (DIG)-labeled λ DNA marker III ( Hin dIII- Eco RI digested, Roche Applied Science) as size marker and 100 picogram mL–1 Cla I- or Bam HI-digested R4pGWB501 plasmid DNA containing P. vittata actin promoter and GUSPlus, both of which can be detected in x-ray film along with experimental samples. .. DNA was transferred to a nylon membrane (GE Healthcare) by capillary blotting ( ) and cross linked by UV irradiation.

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  • 78
    Roche total aegilops tauschii genomic dna
    Substitution (1D/1A) line, 2 n = 42 chromosomes. ( a ) FISH with 5S rDNA (red) and 35S rDNA (green); ( b ) FISH with pAs1 (red) and pSc 119.2 (green). The chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue); ( c ) mc GISH with total genomic <t>DNA</t> from rye—R genome (red), total genomic DNA from Triticum monococcum —A genome (green) and total genomic DNA from Aegilops <t>tauschii</t> —D genome (yellow) with blocking genomic DNA of Aegilops speltoides —B genome (DAPI, blue).
    Total Aegilops Tauschii Genomic Dna, supplied by Roche, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total aegilops tauschii genomic dna/product/Roche
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    total aegilops tauschii genomic dna - by Bioz Stars, 2020-02
    78/100 stars
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    96
    Roche genomic dna
    Flow chart diagrams illustrating genome and transcriptome assembly pipelines. (A) Pipeline for assembly of PGT21 and PGTAus-pan genomes from <t>DNA</t> reads from Australian <t>Pgt</t> isolates. (B) Pipeline for assembly of isolate 21-0 transcriptome from RNA reads from isolated haustoria and germinated spores.
    Genomic Dna, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genomic dna/product/Roche
    Average 96 stars, based on 181 article reviews
    Price from $9.99 to $1999.99
    genomic dna - by Bioz Stars, 2020-02
    96/100 stars
      Buy from Supplier

    Image Search Results


    Substitution (1D/1A) line, 2 n = 42 chromosomes. ( a ) FISH with 5S rDNA (red) and 35S rDNA (green); ( b ) FISH with pAs1 (red) and pSc 119.2 (green). The chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue); ( c ) mc GISH with total genomic DNA from rye—R genome (red), total genomic DNA from Triticum monococcum —A genome (green) and total genomic DNA from Aegilops tauschii —D genome (yellow) with blocking genomic DNA of Aegilops speltoides —B genome (DAPI, blue).

    Journal: International Journal of Molecular Sciences

    Article Title: Molecular, Physicochemical and Rheological Characteristics of Introgressive Triticale/Triticum monococcum ssp. monococcum Lines with Wheat 1D/1A Chromosome Substitution

    doi: 10.3390/ijms140815595

    Figure Lengend Snippet: Substitution (1D/1A) line, 2 n = 42 chromosomes. ( a ) FISH with 5S rDNA (red) and 35S rDNA (green); ( b ) FISH with pAs1 (red) and pSc 119.2 (green). The chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue); ( c ) mc GISH with total genomic DNA from rye—R genome (red), total genomic DNA from Triticum monococcum —A genome (green) and total genomic DNA from Aegilops tauschii —D genome (yellow) with blocking genomic DNA of Aegilops speltoides —B genome (DAPI, blue).

    Article Snippet: Total Aegilops tauschii genomic DNA was used as a probe; it was labeled by nicktranslation with digoxigenin-11-dUTP for the screening, or digoxigenin-11-dUTP and tetramethyl-rhodamine-5-dUTP (Roche) (1:1 ratio) for the identification process.

    Techniques: Fluorescence In Situ Hybridization, Blocking Assay

    Flow chart diagrams illustrating genome and transcriptome assembly pipelines. (A) Pipeline for assembly of PGT21 and PGTAus-pan genomes from DNA reads from Australian Pgt isolates. (B) Pipeline for assembly of isolate 21-0 transcriptome from RNA reads from isolated haustoria and germinated spores.

    Journal: Frontiers in Plant Science

    Article Title: Comparative genomics of Australian isolates of the wheat stem rust pathogen Puccinia graminis f. sp. tritici reveals extensive polymorphism in candidate effector genes

    doi: 10.3389/fpls.2014.00759

    Figure Lengend Snippet: Flow chart diagrams illustrating genome and transcriptome assembly pipelines. (A) Pipeline for assembly of PGT21 and PGTAus-pan genomes from DNA reads from Australian Pgt isolates. (B) Pipeline for assembly of isolate 21-0 transcriptome from RNA reads from isolated haustoria and germinated spores.

    Article Snippet: Pgt isolate 21-0 genomic DNA was sequenced by Roche GS FLX 454 technology at the Australian Genome Research Facility Ltd (AGRF – Australia).

    Techniques: Flow Cytometry, Isolation

    ComEA binds to DNA in vivo . (A) Plasmid-encoded gfp (tat-GFP) or comEA-gfp (tat-ComEA-GFP), both preceded by a tat-signal sequence, were expressed in E. coli . The images shown correspond to the GFP channel, DAPI channel (to visualize DAPI-stained DNA), merged fluorescent images (merge), and phase contrast (Ph). The cells are outlined with dashed lines for tat-ComEA-GFP. Heat-maps showing the fluorescence intensities of the GFP and DAPI signals are depicted for the tat-comEA-gfp expressing cells below the images. (B) ComEA-mCherry aggregation and foci formation after the addition of external DNA. Competence-induced cells without (no DNA) or with external DNA were imaged in the red (mCherry; upper row) or the phase contrast channel (lower row) to visualize ComEA-mCherry localization. The DNA fragments differed in lengths (PCR fragment, 10.3 kb; λDNA, 48.5 kb; gDNA, various lengths). Transforming DNA did not lead to foci formation of periplasmic mCherry alone (preceded by the ComEA signal sequence; ss[ComEA]-mCherry). (C) Colocalization (merged image) of ComEA-mCherry (red channel) and YoYo-1-stained transforming DNA (green channel). The outline of the cells is shown in the phase contrast image (Ph). Scale bars in all images, 2 µm. (D) DNA uptake requires ComEA. DNA uptake of competent V. cholerae cells was tested using a whole-cell duplex PCR assay. All mutant strains were tested in a comEC positive (+) and negative (−) background. The lower PCR fragments indicate acceptor strain DNA (gDNA, acceptor); the upper band indicates internalized transforming DNA (tDNA). L, ladder.

    Journal: PLoS Genetics

    Article Title: ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

    doi: 10.1371/journal.pgen.1004066

    Figure Lengend Snippet: ComEA binds to DNA in vivo . (A) Plasmid-encoded gfp (tat-GFP) or comEA-gfp (tat-ComEA-GFP), both preceded by a tat-signal sequence, were expressed in E. coli . The images shown correspond to the GFP channel, DAPI channel (to visualize DAPI-stained DNA), merged fluorescent images (merge), and phase contrast (Ph). The cells are outlined with dashed lines for tat-ComEA-GFP. Heat-maps showing the fluorescence intensities of the GFP and DAPI signals are depicted for the tat-comEA-gfp expressing cells below the images. (B) ComEA-mCherry aggregation and foci formation after the addition of external DNA. Competence-induced cells without (no DNA) or with external DNA were imaged in the red (mCherry; upper row) or the phase contrast channel (lower row) to visualize ComEA-mCherry localization. The DNA fragments differed in lengths (PCR fragment, 10.3 kb; λDNA, 48.5 kb; gDNA, various lengths). Transforming DNA did not lead to foci formation of periplasmic mCherry alone (preceded by the ComEA signal sequence; ss[ComEA]-mCherry). (C) Colocalization (merged image) of ComEA-mCherry (red channel) and YoYo-1-stained transforming DNA (green channel). The outline of the cells is shown in the phase contrast image (Ph). Scale bars in all images, 2 µm. (D) DNA uptake requires ComEA. DNA uptake of competent V. cholerae cells was tested using a whole-cell duplex PCR assay. All mutant strains were tested in a comEC positive (+) and negative (−) background. The lower PCR fragments indicate acceptor strain DNA (gDNA, acceptor); the upper band indicates internalized transforming DNA (tDNA). L, ladder.

    Article Snippet: A total of 50 µl of washed culture was mixed with 1 µg of either gDNA derived from V. cholerae strain A1552-lacZ-Kan , commercially available phage lambda DNA (Roche) or a 10.3 kb fragment amplified through PCR.

    Techniques: In Vivo, Plasmid Preparation, Sequencing, Staining, Fluorescence, Expressing, Polymerase Chain Reaction, Mutagenesis

    Localization of the ComEA protein in naturally competent V. cholerae cells. (A) Expression and distribution of ComEA-mCherry (upper row) or signal sequence[ComEA] (amino acids 1–25)-mCherry fusion proteins (lower row) within competent V. cholerae cells. Fluorescent signals for mCherry or DAPI-stained genomic DNA were visualized and compared with each other (merge) and the corresponding phase contrast image (Ph). (B) Representative fluorescence loss in photobleaching (FLIP) experiment to demonstrate the degree of mobility of ComEA-mCherry in live bacteria. Bleaching of the region-of-interest (ROI) 1 (indicated as 1 in the images on the right) was initiated after the acquisition of 20 frames and repeated after every frame. The fluorescence intensities of ROIs 1–3 were measured for a total of 20 sec and normalized to the average fluorescence intensity of the first 10 frames. The moving averages (period n = 5) are indicated with black lines. The average fluorescence intensity projections before (pre-bleach) and after bleaching (post-bleach) are shown on the right. Scale bars, 2 µm.

    Journal: PLoS Genetics

    Article Title: ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

    doi: 10.1371/journal.pgen.1004066

    Figure Lengend Snippet: Localization of the ComEA protein in naturally competent V. cholerae cells. (A) Expression and distribution of ComEA-mCherry (upper row) or signal sequence[ComEA] (amino acids 1–25)-mCherry fusion proteins (lower row) within competent V. cholerae cells. Fluorescent signals for mCherry or DAPI-stained genomic DNA were visualized and compared with each other (merge) and the corresponding phase contrast image (Ph). (B) Representative fluorescence loss in photobleaching (FLIP) experiment to demonstrate the degree of mobility of ComEA-mCherry in live bacteria. Bleaching of the region-of-interest (ROI) 1 (indicated as 1 in the images on the right) was initiated after the acquisition of 20 frames and repeated after every frame. The fluorescence intensities of ROIs 1–3 were measured for a total of 20 sec and normalized to the average fluorescence intensity of the first 10 frames. The moving averages (period n = 5) are indicated with black lines. The average fluorescence intensity projections before (pre-bleach) and after bleaching (post-bleach) are shown on the right. Scale bars, 2 µm.

    Article Snippet: A total of 50 µl of washed culture was mixed with 1 µg of either gDNA derived from V. cholerae strain A1552-lacZ-Kan , commercially available phage lambda DNA (Roche) or a 10.3 kb fragment amplified through PCR.

    Techniques: Expressing, Sequencing, Staining, Fluorescence, Size-exclusion Chromatography

    Generation and validation of a Casp2 C320S mice. ( a ) A representative DNA agarose gel image from genotyping of WT, Casp2 +/C320S (heterozygous) and Casp2 C320S/C320S (homozygous) mice. The size of expected DNA fragments generated per genotype are indicated (bp). ( b ) DNA sequencing confirmation of the different genotypes as indicated. The red and blue arrows indicate the point mutation sites (G, G/C or C) and the silent mutation site (T, T/A or A), respectively. ( c ) Immunoblot analysis of caspase-2 expression in splenocytes from WT and Casp2 C320S/C320S mice. β-actin is shown as loading control. ( d ) Genotype frequencies of the progeny from Casp2 +/C320S (heterozygous) intercrossing including observed and expected numbers of offspring. Observed and expected frequency (%) is indicated in parentheses. Statistical comparison was performed by χ 2 test.

    Journal: Oncogene

    Article Title: Caspase-2-mediated cell death is required for deleting aneuploid cells

    doi: 10.1038/onc.2016.423

    Figure Lengend Snippet: Generation and validation of a Casp2 C320S mice. ( a ) A representative DNA agarose gel image from genotyping of WT, Casp2 +/C320S (heterozygous) and Casp2 C320S/C320S (homozygous) mice. The size of expected DNA fragments generated per genotype are indicated (bp). ( b ) DNA sequencing confirmation of the different genotypes as indicated. The red and blue arrows indicate the point mutation sites (G, G/C or C) and the silent mutation site (T, T/A or A), respectively. ( c ) Immunoblot analysis of caspase-2 expression in splenocytes from WT and Casp2 C320S/C320S mice. β-actin is shown as loading control. ( d ) Genotype frequencies of the progeny from Casp2 +/C320S (heterozygous) intercrossing including observed and expected numbers of offspring. Observed and expected frequency (%) is indicated in parentheses. Statistical comparison was performed by χ 2 test.

    Article Snippet: For genotyping of mutant mice including founder mice, genomic DNA was extracted from postnatal day 10 tail tissue using High Pure PCR Template Preparation Kit (ROCHE, Basel Schweiz, Switzerland).

    Techniques: Mouse Assay, Agarose Gel Electrophoresis, Generated, DNA Sequencing, Mutagenesis, Expressing