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TaKaRa pcr fragment
Identification of transgenic Arabidopsis plants and their salt tolerance phenotypes. ( A ) Identification of transgenes by <t>PCR</t> amplification of genomic DNA using <t>drnf1</t> gene-specific primers; ( B ) Semi-quantitative RT-PCR analysis of drnf1 transcription. M: B031 DNA ladder (Dingguo, Beijing, China). WT: wild-type plant. 35d1-6: independent transgenic lines; ( C ) Germination performance of transgenic seeds on solid media supplemented with various concentrations of NaCl; ( D ) Growth performance of transgenic plants in 150 mM NaCl solution-watered soils. Three-week-old plantlets (0 d) were subjected to salt stress treatment.
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1) Product Images from "The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant"

Article Title: The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant

Journal: Genes

doi: 10.3390/genes9090441

Identification of transgenic Arabidopsis plants and their salt tolerance phenotypes. ( A ) Identification of transgenes by PCR amplification of genomic DNA using drnf1 gene-specific primers; ( B ) Semi-quantitative RT-PCR analysis of drnf1 transcription. M: B031 DNA ladder (Dingguo, Beijing, China). WT: wild-type plant. 35d1-6: independent transgenic lines; ( C ) Germination performance of transgenic seeds on solid media supplemented with various concentrations of NaCl; ( D ) Growth performance of transgenic plants in 150 mM NaCl solution-watered soils. Three-week-old plantlets (0 d) were subjected to salt stress treatment.
Figure Legend Snippet: Identification of transgenic Arabidopsis plants and their salt tolerance phenotypes. ( A ) Identification of transgenes by PCR amplification of genomic DNA using drnf1 gene-specific primers; ( B ) Semi-quantitative RT-PCR analysis of drnf1 transcription. M: B031 DNA ladder (Dingguo, Beijing, China). WT: wild-type plant. 35d1-6: independent transgenic lines; ( C ) Germination performance of transgenic seeds on solid media supplemented with various concentrations of NaCl; ( D ) Growth performance of transgenic plants in 150 mM NaCl solution-watered soils. Three-week-old plantlets (0 d) were subjected to salt stress treatment.

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR

Identification of transgenic drnf1 Synechocystis and its resistance to short-term salt stress. ( A ) Identification of the transgenic strain using drnf1 gene-specific primers by PCR. WT: wild-type strain. drnf1 -O: transgenic strain. M: DNA marker; ( B ) Semi-quantitative RT-PCR detection of drnf1 transcription in the transgenic strain; ( C ) Changes of relative Fv/Fm during salt stresses for 3 h. Data are mean ± s.d. ( n = 5).
Figure Legend Snippet: Identification of transgenic drnf1 Synechocystis and its resistance to short-term salt stress. ( A ) Identification of the transgenic strain using drnf1 gene-specific primers by PCR. WT: wild-type strain. drnf1 -O: transgenic strain. M: DNA marker; ( B ) Semi-quantitative RT-PCR detection of drnf1 transcription in the transgenic strain; ( C ) Changes of relative Fv/Fm during salt stresses for 3 h. Data are mean ± s.d. ( n = 5).

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Marker, Quantitative RT-PCR

Salt stress response of Nostoc flagelliforme and induction of drnf1 transcription. ( A ) Physiologically recovered samples were treated by NaCl solutions for 27 h and then transferred to normal solutions for physiological recovery for 20 h. Physiological changes were indexed with the Fv/Fm parameter. Data are mean ± s.d. ( n = 5); ( B ) Semi-quantitative RT-PCR analysis of transcription levels of drnf1 gene under 0.4 M NaCl for different times. 16S rRNA gene was used an internal reference.
Figure Legend Snippet: Salt stress response of Nostoc flagelliforme and induction of drnf1 transcription. ( A ) Physiologically recovered samples were treated by NaCl solutions for 27 h and then transferred to normal solutions for physiological recovery for 20 h. Physiological changes were indexed with the Fv/Fm parameter. Data are mean ± s.d. ( n = 5); ( B ) Semi-quantitative RT-PCR analysis of transcription levels of drnf1 gene under 0.4 M NaCl for different times. 16S rRNA gene was used an internal reference.

Techniques Used: Quantitative RT-PCR

2) Product Images from "Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9"

Article Title: Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E19-04-0223

CRISPR/Cas9-mediated knock-out of Syp and PSD-95 to examine the correlation of endogenous expression of pre- and postsynaptic proteins in cultured neurons. (A, C) The structure of the rat Syp locus and the nucleotide sequence of exon 4–5 (A), and the structure of rat PSD-95 locus and the nucleotide sequence of exon 3–8 (C). Intron sequences are shown in gray. The gRNA targeting sequences are underlined, and the PAM sequences are shown in bold. The predicted Cas9-gRNA cutting positions are indicated with red triangles. Arrows denote the position of the PCR primers used for genotyping. (B, D) Genomic PCR analysis of primary neuronal cultures nucleofected with the CRISPR constructs. Primary hippocampal neurons were nucleofected with CAG-EGFP and the following plasmids: CBh-Cas9 without gRNA (B and D, lane 1), a combination of CBh-Cas9/U6-gRNA (Syp#1) and CBh-Cas9/U6-gRNA (Syp#4) (B, lane 2), a combination of CBh-Cas9/U6-gRNA (Syp#2) and CBh-Cas9/U6-gRNA (Syp#3) (B, lane 3), a combination of CBh-Cas9/U6-gRNA (PSD-95#1) and CBh-Cas9/U6-gRNA (PSD-95#2) (D, lane 2), or a combination of CBh-Cas9/U6-gRNA (PSD-95#1) and CBh-Cas9/U6-gRNA (PSD-95#3) (D, lane 3). DNAs were extracted from the cultured neurons at 19 DIV and subjected to PCR analysis. Targeted genome modifications were also verified by Sanger sequencing of the PCR amplicons (see Supplemental Figures S3 and S4). (E, F) Western blot analysis of whole-cell lysates from 19 DIV rat primary hippocampal neurons nucleofected with the indicated CRISPR constructs. Immunoblots were probed with indicated antibodies, and densitometric analysis of blots was performed to determine the fold differences in endogenous expression of PSD-95 (E) or Syp (F). The graphs show quantitative densitometry and statistical analysis of the Western blot bands; n = 3 (three blot membranes from three sample sets); mean ± SEM (error bars); *p
Figure Legend Snippet: CRISPR/Cas9-mediated knock-out of Syp and PSD-95 to examine the correlation of endogenous expression of pre- and postsynaptic proteins in cultured neurons. (A, C) The structure of the rat Syp locus and the nucleotide sequence of exon 4–5 (A), and the structure of rat PSD-95 locus and the nucleotide sequence of exon 3–8 (C). Intron sequences are shown in gray. The gRNA targeting sequences are underlined, and the PAM sequences are shown in bold. The predicted Cas9-gRNA cutting positions are indicated with red triangles. Arrows denote the position of the PCR primers used for genotyping. (B, D) Genomic PCR analysis of primary neuronal cultures nucleofected with the CRISPR constructs. Primary hippocampal neurons were nucleofected with CAG-EGFP and the following plasmids: CBh-Cas9 without gRNA (B and D, lane 1), a combination of CBh-Cas9/U6-gRNA (Syp#1) and CBh-Cas9/U6-gRNA (Syp#4) (B, lane 2), a combination of CBh-Cas9/U6-gRNA (Syp#2) and CBh-Cas9/U6-gRNA (Syp#3) (B, lane 3), a combination of CBh-Cas9/U6-gRNA (PSD-95#1) and CBh-Cas9/U6-gRNA (PSD-95#2) (D, lane 2), or a combination of CBh-Cas9/U6-gRNA (PSD-95#1) and CBh-Cas9/U6-gRNA (PSD-95#3) (D, lane 3). DNAs were extracted from the cultured neurons at 19 DIV and subjected to PCR analysis. Targeted genome modifications were also verified by Sanger sequencing of the PCR amplicons (see Supplemental Figures S3 and S4). (E, F) Western blot analysis of whole-cell lysates from 19 DIV rat primary hippocampal neurons nucleofected with the indicated CRISPR constructs. Immunoblots were probed with indicated antibodies, and densitometric analysis of blots was performed to determine the fold differences in endogenous expression of PSD-95 (E) or Syp (F). The graphs show quantitative densitometry and statistical analysis of the Western blot bands; n = 3 (three blot membranes from three sample sets); mean ± SEM (error bars); *p

Techniques Used: CRISPR, Knock-Out, Expressing, Cell Culture, Sequencing, Polymerase Chain Reaction, Construct, Western Blot

Validation of CRISPR/Cas9-mediated knock-in of fluorescent genes into cultured neurons. (A, F) Structures of the rat PSD-95 (A) and Syp (F) loci and the knock-in constructs to produce EGFP and mCherry fusion proteins. The gRNA targeting sequences are underlined, and the PAM sequences are shown in red. The predicted Cas9-gRNA cutting positions are indicated with a scissor-cutting symbols. These two gRNAs for each gene were used together to increase the knock-in efficiency. Locations of genotyping primers sets to detect EGFP or mCherry knock-in alleles of PSD-95 (EGFP: rPsd-F1 and EGFP-R1; mCherry: rPSD-F1 and mCherry-R1) and Syp (EGFP: rSyp-F1 and EGFP-R1; mCherry: rSyp-F1 and mCherry-R1) are shown. (B–E) Genomic PCR analysis of nucleofected neurons. Cortical (B, D) or hippocampal (C, E) neurons were nucleofected with three plasmids: PSD-95 targeting vector (EGFP), CAG-mCherry, and CBh-Cas9 with or without gRNAs for PSD-95 (B, C), or PSD-95 targeting vector (mCherry), CAG-EGFP, and CBh-Cas9 with or without gRNAs for PSD-95 (D, E). Genomic DNA samples extracted from the cultured neurons at 21 DIV were subjected to PCR analysis. PCR primer sets were used to detect the fluorescent protein knock-in alleles, transfected targeting vectors, and endogenous rat Actb , respectively. Agarose gel electrophoresis of PCR products are shown: lane 1, DNA prepared from the primary neuron cultures nucleofected without CBh-Cas9/U6-gRNA (PSD-95); lane 2, DNA prepared from the neuron primary cultures nucleofected with CBh-Cas9/U6-gRNA (PSD-95); lane 3, wild-type rat genomic DNA; lane 4, PSD-95 targeting vector plasmid DNA. (G–J) Genomic PCR analysis of nucleofected neurons. Cortical (G, I) or hippocampal (H, J) neurons were nucleofected with three plasmids: Syp targeting vector (EGFP), CAG-mCherry, and CBh-Cas9 with or without gRNAs for Syp (G, H), or Syp targeting vector (mCherry), CAG-EGFP, and CBh-Cas9 with or without gRNAs for Syp (I, J). Genomic DNA samples extracted from the cultured neurons at 21 DIV were subjected to PCR analysis. PCR primer sets were used to detect the fluorescent protein knock-in alleles, transfected targeting vectors, and endogenous rat Actb , respectively. Agarose gel electrophoresis of PCR products are shown: lane 1, DNA prepared from the primary neuron cultures nucleofected without CBh-Cas9/U6-gRNA (Syp); lane 2, DNA prepared from the neuron primary cultures nucleofected with CBh-Cas9/U6-gRNA (Syp); lane 3, wild-type rat genomic DNA; lane 4, Syp targeting vector plasmid DNA.
Figure Legend Snippet: Validation of CRISPR/Cas9-mediated knock-in of fluorescent genes into cultured neurons. (A, F) Structures of the rat PSD-95 (A) and Syp (F) loci and the knock-in constructs to produce EGFP and mCherry fusion proteins. The gRNA targeting sequences are underlined, and the PAM sequences are shown in red. The predicted Cas9-gRNA cutting positions are indicated with a scissor-cutting symbols. These two gRNAs for each gene were used together to increase the knock-in efficiency. Locations of genotyping primers sets to detect EGFP or mCherry knock-in alleles of PSD-95 (EGFP: rPsd-F1 and EGFP-R1; mCherry: rPSD-F1 and mCherry-R1) and Syp (EGFP: rSyp-F1 and EGFP-R1; mCherry: rSyp-F1 and mCherry-R1) are shown. (B–E) Genomic PCR analysis of nucleofected neurons. Cortical (B, D) or hippocampal (C, E) neurons were nucleofected with three plasmids: PSD-95 targeting vector (EGFP), CAG-mCherry, and CBh-Cas9 with or without gRNAs for PSD-95 (B, C), or PSD-95 targeting vector (mCherry), CAG-EGFP, and CBh-Cas9 with or without gRNAs for PSD-95 (D, E). Genomic DNA samples extracted from the cultured neurons at 21 DIV were subjected to PCR analysis. PCR primer sets were used to detect the fluorescent protein knock-in alleles, transfected targeting vectors, and endogenous rat Actb , respectively. Agarose gel electrophoresis of PCR products are shown: lane 1, DNA prepared from the primary neuron cultures nucleofected without CBh-Cas9/U6-gRNA (PSD-95); lane 2, DNA prepared from the neuron primary cultures nucleofected with CBh-Cas9/U6-gRNA (PSD-95); lane 3, wild-type rat genomic DNA; lane 4, PSD-95 targeting vector plasmid DNA. (G–J) Genomic PCR analysis of nucleofected neurons. Cortical (G, I) or hippocampal (H, J) neurons were nucleofected with three plasmids: Syp targeting vector (EGFP), CAG-mCherry, and CBh-Cas9 with or without gRNAs for Syp (G, H), or Syp targeting vector (mCherry), CAG-EGFP, and CBh-Cas9 with or without gRNAs for Syp (I, J). Genomic DNA samples extracted from the cultured neurons at 21 DIV were subjected to PCR analysis. PCR primer sets were used to detect the fluorescent protein knock-in alleles, transfected targeting vectors, and endogenous rat Actb , respectively. Agarose gel electrophoresis of PCR products are shown: lane 1, DNA prepared from the primary neuron cultures nucleofected without CBh-Cas9/U6-gRNA (Syp); lane 2, DNA prepared from the neuron primary cultures nucleofected with CBh-Cas9/U6-gRNA (Syp); lane 3, wild-type rat genomic DNA; lane 4, Syp targeting vector plasmid DNA.

Techniques Used: CRISPR, Knock-In, Cell Culture, Construct, Polymerase Chain Reaction, Plasmid Preparation, Transfection, Agarose Gel Electrophoresis

3) Product Images from "The role of base excision repair genes OGG1, APN1 and APN2 in benzo[a]pyrene-7,8-dione induced p53 mutagenesis"

Article Title: The role of base excision repair genes OGG1, APN1 and APN2 in benzo[a]pyrene-7,8-dione induced p53 mutagenesis

Journal: Mutation research

doi: 10.1016/j.mrgentox.2012.10.003

The yeast p53 assay and real-time PCR confirming the mutant yeast strains. A) Schematic diagram showing the assay used to determine p53 mutant frequency (based on red/white selection), mutation pattern and mutation spectrum in wild type ( yIG397 ), ogg1
Figure Legend Snippet: The yeast p53 assay and real-time PCR confirming the mutant yeast strains. A) Schematic diagram showing the assay used to determine p53 mutant frequency (based on red/white selection), mutation pattern and mutation spectrum in wild type ( yIG397 ), ogg1

Techniques Used: Real-time Polymerase Chain Reaction, Mutagenesis, Selection

4) Product Images from "A 40 kDa protein of the inner membrane is the mitochondrial calcium uniporter"

Article Title: A 40 kDa protein of the inner membrane is the mitochondrial calcium uniporter

Journal: Nature

doi: 10.1038/nature10230

MCU includes two highly conserved transmembrane domains and is ubiquitously expressed in mammals, similarly to its putative regulator MICU1 a , Alignment of the putative transmembrane domain and pore region of MCU proteins from 14 different species. b , Quantitative Real Time PCR analysis of mouse tissues. mRNA extraction and quantitative PCR as described in the methods section. Expression levels are normalized to skeletal muscle, and presented as means ± S.D. (n = 3).
Figure Legend Snippet: MCU includes two highly conserved transmembrane domains and is ubiquitously expressed in mammals, similarly to its putative regulator MICU1 a , Alignment of the putative transmembrane domain and pore region of MCU proteins from 14 different species. b , Quantitative Real Time PCR analysis of mouse tissues. mRNA extraction and quantitative PCR as described in the methods section. Expression levels are normalized to skeletal muscle, and presented as means ± S.D. (n = 3).

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

5) Product Images from "Phosphatidylserine Synthase Controls Cell Elongation Especially in the Uppermost Internode in Rice by Regulation of Exocytosis"

Article Title: Phosphatidylserine Synthase Controls Cell Elongation Especially in the Uppermost Internode in Rice by Regulation of Exocytosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0153119

Positional cloning of OsPSS-1 . (A) Fine mapping of the OsPSS-1 locus. Molecular markers and number of recombinants (recs) are shown. BAC, bacterial artificial chromosome; ORF, open reading frame. (B) and (C) The WT genomic segment of OsPSS-1 completely rescues plant stature. L1 and L2 denote plants from T1 transgenic lines. In (C), the nodes of the sui1-4 mutant and L1 plants are indicated with white arrowheads. Scale bars: 10 cm. (D) OsPSS-1- RNAi transgenic lines mimic the phenotype of sui1-4 plants. R1, R2, and R3 represent three independent T1 transgenic lines. Scale bar: 10 cm. (E) Internodes in WT, R1, R2, and R3 plants; white arrowheads indicate the nodes. Scale bar: 10 cm. (F) qRT-PCR reveals lower OsPSS-1 expression in mutant plants than in WT plants, but transcript levels of OsPSS-1 homologs (LOC_Os05g48060 and LOC_Os01g49024) were not affected. Values are means ± standard error of three independent experiments. Significant differences were identified with Student’s t -test (**p
Figure Legend Snippet: Positional cloning of OsPSS-1 . (A) Fine mapping of the OsPSS-1 locus. Molecular markers and number of recombinants (recs) are shown. BAC, bacterial artificial chromosome; ORF, open reading frame. (B) and (C) The WT genomic segment of OsPSS-1 completely rescues plant stature. L1 and L2 denote plants from T1 transgenic lines. In (C), the nodes of the sui1-4 mutant and L1 plants are indicated with white arrowheads. Scale bars: 10 cm. (D) OsPSS-1- RNAi transgenic lines mimic the phenotype of sui1-4 plants. R1, R2, and R3 represent three independent T1 transgenic lines. Scale bar: 10 cm. (E) Internodes in WT, R1, R2, and R3 plants; white arrowheads indicate the nodes. Scale bar: 10 cm. (F) qRT-PCR reveals lower OsPSS-1 expression in mutant plants than in WT plants, but transcript levels of OsPSS-1 homologs (LOC_Os05g48060 and LOC_Os01g49024) were not affected. Values are means ± standard error of three independent experiments. Significant differences were identified with Student’s t -test (**p

Techniques Used: Clone Assay, BAC Assay, Transgenic Assay, Mutagenesis, Quantitative RT-PCR, Expressing

OsPSS-1 expression and histochemical staining of pOsPSS-1 :: GUS transgenic plants. (A) qRT-PCR shows that OsPSS-1 is ubiquitously expressed in various organs, with the highest level in panicles. For each organ, the sui1-4 mutant displayed lower expression levels than WT plants. Values are means ± standard error of three independent experiments. Significant differences were identified with Student’s t -test (**p
Figure Legend Snippet: OsPSS-1 expression and histochemical staining of pOsPSS-1 :: GUS transgenic plants. (A) qRT-PCR shows that OsPSS-1 is ubiquitously expressed in various organs, with the highest level in panicles. For each organ, the sui1-4 mutant displayed lower expression levels than WT plants. Values are means ± standard error of three independent experiments. Significant differences were identified with Student’s t -test (**p

Techniques Used: Expressing, Staining, Transgenic Assay, Quantitative RT-PCR, Mutagenesis

6) Product Images from "Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria"

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

Journal: Nature Communications

doi: 10.1038/s41467-020-16731-6

Potential use of CapsidCas13a for bacterial gene detection. a A schematic illustration of generation of PICI-based EC-CapsidCas13a targeting bla IMP-1 . Mitomycin C induction promotes the packaging of PICImid carrying a CRISPR-Cas13a system and kanamycin (Km) resistance gene into the capsid of helper phage Φ80. b – d Bactericidal activity test of PICI-based EC-CapsidCas13a::KanR- bla IMP-1 (in tenfold serial dilutions) against E. coli MC1061 with or without the expression of target gene was carried out on LB agar plates ( b ); and the test results were judged by observing bacterial growth on LB bottom agar plates supplemented with Km ( c ), or observation of cell lysis on drug-free LB bottom agar plates ( d ); noting that the former assay had an enhanced sensitivity by about three orders of magnitude against the bacteria carrying target gene. e – j The PICI-based EC-CapsidCas13a(s) were applicable to detect various carbapenem resistance genes ( bla IMP-1 , bla OXA-48 , and bla VIM-2 ) regardless of their location on either the plasmid or chromosome ( e , f ), whereas EC-CapsidCas9 could detect genes located on the chromosome but not on the plasmid ( g ); and the PICI-based EC-CapsidCas13a(s) also effectively detected toxin-encoded genes ( h ), differentiated different genes located on the same plasmid ( i ), and were also applicable to clinical isolates ( j , left panel) as being verified by PCR ( j , right panel). k SA-CapsidCas13a::TetR- mecA generated by packaging mecA -targeting CRISPR-Cas13a into capsid of S. aureus phage 80α exhibited mecA -specific bactericidal activity against MRSA, but not S. aureus strains deficient in mecA . All assays were replicated three times. Uncropped images of the gels are available in the Supplementary Information.
Figure Legend Snippet: Potential use of CapsidCas13a for bacterial gene detection. a A schematic illustration of generation of PICI-based EC-CapsidCas13a targeting bla IMP-1 . Mitomycin C induction promotes the packaging of PICImid carrying a CRISPR-Cas13a system and kanamycin (Km) resistance gene into the capsid of helper phage Φ80. b – d Bactericidal activity test of PICI-based EC-CapsidCas13a::KanR- bla IMP-1 (in tenfold serial dilutions) against E. coli MC1061 with or without the expression of target gene was carried out on LB agar plates ( b ); and the test results were judged by observing bacterial growth on LB bottom agar plates supplemented with Km ( c ), or observation of cell lysis on drug-free LB bottom agar plates ( d ); noting that the former assay had an enhanced sensitivity by about three orders of magnitude against the bacteria carrying target gene. e – j The PICI-based EC-CapsidCas13a(s) were applicable to detect various carbapenem resistance genes ( bla IMP-1 , bla OXA-48 , and bla VIM-2 ) regardless of their location on either the plasmid or chromosome ( e , f ), whereas EC-CapsidCas9 could detect genes located on the chromosome but not on the plasmid ( g ); and the PICI-based EC-CapsidCas13a(s) also effectively detected toxin-encoded genes ( h ), differentiated different genes located on the same plasmid ( i ), and were also applicable to clinical isolates ( j , left panel) as being verified by PCR ( j , right panel). k SA-CapsidCas13a::TetR- mecA generated by packaging mecA -targeting CRISPR-Cas13a into capsid of S. aureus phage 80α exhibited mecA -specific bactericidal activity against MRSA, but not S. aureus strains deficient in mecA . All assays were replicated three times. Uncropped images of the gels are available in the Supplementary Information.

Techniques Used: CRISPR, Activity Assay, Expressing, Lysis, Bla VIM Assay, Plasmid Preparation, Polymerase Chain Reaction, Generated

7) Product Images from "Centrin3 in trypanosomes maintains the stability of a flagellar inner-arm dynein for cell motility"

Article Title: Centrin3 in trypanosomes maintains the stability of a flagellar inner-arm dynein for cell motility

Journal: Nature communications

doi: 10.1038/ncomms5060

RNAi of TbCentrin3 in procyclic trypanosomes impairs cell motility ( A ). TbCentrin3 mRNA level in non-induced control cells and RNAi cells detected by quantitative RT-PCR. Three independent experiments were carried out and the error bars represent S.D. ( B ). TbCentrin3::PTP protein level in control and RNAi cells detected by western blotting with anti-Protein A antibody. Level of TbPSA6, the α6 subunit of the 26S proteasome, was included as the loading control. ( C ). RNAi of TbCentrin3 slowed down cell proliferation. ( D ). Sedimentation assays to monitor cell motility. The parental 29-13 strain, the non-induced control, and RNAi cells after tetracycline induction for 3 days were incubated in cuvettes, and the cell density (optical density) was determined and plotted against the time of incubation (hours). Three independent experiments were carried out and the error bars represent S.D. ( E, F ). Tracing the motility of non-induced control cells and TbCentrin3 RNAi cells (day 3) by video microscopy. Black arrows indicate the posterior tip of the cells, and the white dashed lines show the posterior of cells at the start of time-lapse video microscopy. Bar: 10 µm. Pie charts inserted in panel F show the percentage of cells scored as runner (white), tumbler (gray) or immotile (black) in non-induced control and TbCentrin3 RNAi cells. n: number of cells counted.
Figure Legend Snippet: RNAi of TbCentrin3 in procyclic trypanosomes impairs cell motility ( A ). TbCentrin3 mRNA level in non-induced control cells and RNAi cells detected by quantitative RT-PCR. Three independent experiments were carried out and the error bars represent S.D. ( B ). TbCentrin3::PTP protein level in control and RNAi cells detected by western blotting with anti-Protein A antibody. Level of TbPSA6, the α6 subunit of the 26S proteasome, was included as the loading control. ( C ). RNAi of TbCentrin3 slowed down cell proliferation. ( D ). Sedimentation assays to monitor cell motility. The parental 29-13 strain, the non-induced control, and RNAi cells after tetracycline induction for 3 days were incubated in cuvettes, and the cell density (optical density) was determined and plotted against the time of incubation (hours). Three independent experiments were carried out and the error bars represent S.D. ( E, F ). Tracing the motility of non-induced control cells and TbCentrin3 RNAi cells (day 3) by video microscopy. Black arrows indicate the posterior tip of the cells, and the white dashed lines show the posterior of cells at the start of time-lapse video microscopy. Bar: 10 µm. Pie charts inserted in panel F show the percentage of cells scored as runner (white), tumbler (gray) or immotile (black) in non-induced control and TbCentrin3 RNAi cells. n: number of cells counted.

Techniques Used: Quantitative RT-PCR, Western Blot, Sedimentation, Incubation, Microscopy

8) Product Images from "Engineering of Fatty Acid Synthases (FASs) to Boost the Production of Medium-Chain Fatty Acids (MCFAs) in Mucor circinelloides"

Article Title: Engineering of Fatty Acid Synthases (FASs) to Boost the Production of Medium-Chain Fatty Acids (MCFAs) in Mucor circinelloides

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20030786

( A , B ) Expression of TE-01 , TE-02 , TE-03 , and TE-04 genes. ( A ) Structure of plasmids pMAT1552-pyrF-TE-01, pMAT1552-pyrF-TE-02, pMAT1552-pyrF-TE-03, pMAT1552-pyrF-TE-04 for TE-01 , TE-02 , TE-03 , TE-04 genes over-expressing in M. circinelloides M65 are demonstrated. Green boxes indicate the coding region of thioesterase genes. ( B ) Polymerase chain reaction (PCR) amplification of genome of control strain (A) and thioesterase gene (TE) over-expressing strains i.e., M65-TE-01, M65-TE-02, M65-TE-03, and M65-TE-04 was demonstrated to as B–E, respectively with the primers ( Supplementary materials: Table S2 ). M, Gene Ruler DNA Ladder Mix. Sizes in kb of the relevant maker fragments are indicated.
Figure Legend Snippet: ( A , B ) Expression of TE-01 , TE-02 , TE-03 , and TE-04 genes. ( A ) Structure of plasmids pMAT1552-pyrF-TE-01, pMAT1552-pyrF-TE-02, pMAT1552-pyrF-TE-03, pMAT1552-pyrF-TE-04 for TE-01 , TE-02 , TE-03 , TE-04 genes over-expressing in M. circinelloides M65 are demonstrated. Green boxes indicate the coding region of thioesterase genes. ( B ) Polymerase chain reaction (PCR) amplification of genome of control strain (A) and thioesterase gene (TE) over-expressing strains i.e., M65-TE-01, M65-TE-02, M65-TE-03, and M65-TE-04 was demonstrated to as B–E, respectively with the primers ( Supplementary materials: Table S2 ). M, Gene Ruler DNA Ladder Mix. Sizes in kb of the relevant maker fragments are indicated.

Techniques Used: Expressing, Polymerase Chain Reaction, Amplification

9) Product Images from "The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant"

Article Title: The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant

Journal: Genes

doi: 10.3390/genes9090441

Identification of transgenic Arabidopsis plants and their salt tolerance phenotypes. ( A ) Identification of transgenes by PCR amplification of genomic DNA using drnf1 gene-specific primers; ( B ) Semi-quantitative RT-PCR analysis of drnf1 transcription. M: B031 DNA ladder (Dingguo, Beijing, China). WT: wild-type plant. 35d1-6: independent transgenic lines; ( C ) Germination performance of transgenic seeds on solid media supplemented with various concentrations of NaCl; ( D ) Growth performance of transgenic plants in 150 mM NaCl solution-watered soils. Three-week-old plantlets (0 d) were subjected to salt stress treatment.
Figure Legend Snippet: Identification of transgenic Arabidopsis plants and their salt tolerance phenotypes. ( A ) Identification of transgenes by PCR amplification of genomic DNA using drnf1 gene-specific primers; ( B ) Semi-quantitative RT-PCR analysis of drnf1 transcription. M: B031 DNA ladder (Dingguo, Beijing, China). WT: wild-type plant. 35d1-6: independent transgenic lines; ( C ) Germination performance of transgenic seeds on solid media supplemented with various concentrations of NaCl; ( D ) Growth performance of transgenic plants in 150 mM NaCl solution-watered soils. Three-week-old plantlets (0 d) were subjected to salt stress treatment.

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR

Identification of transgenic drnf1 Synechocystis and its resistance to short-term salt stress. ( A ) Identification of the transgenic strain using drnf1 gene-specific primers by PCR. WT: wild-type strain. drnf1 -O: transgenic strain. M: DNA marker; ( B ) Semi-quantitative RT-PCR detection of drnf1 transcription in the transgenic strain; ( C ) Changes of relative Fv/Fm during salt stresses for 3 h. Data are mean ± s.d. ( n = 5).
Figure Legend Snippet: Identification of transgenic drnf1 Synechocystis and its resistance to short-term salt stress. ( A ) Identification of the transgenic strain using drnf1 gene-specific primers by PCR. WT: wild-type strain. drnf1 -O: transgenic strain. M: DNA marker; ( B ) Semi-quantitative RT-PCR detection of drnf1 transcription in the transgenic strain; ( C ) Changes of relative Fv/Fm during salt stresses for 3 h. Data are mean ± s.d. ( n = 5).

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Marker, Quantitative RT-PCR

Salt stress response of Nostoc flagelliforme and induction of drnf1 transcription. ( A ) Physiologically recovered samples were treated by NaCl solutions for 27 h and then transferred to normal solutions for physiological recovery for 20 h. Physiological changes were indexed with the Fv/Fm parameter. Data are mean ± s.d. ( n = 5); ( B ) Semi-quantitative RT-PCR analysis of transcription levels of drnf1 gene under 0.4 M NaCl for different times. 16S rRNA gene was used an internal reference.
Figure Legend Snippet: Salt stress response of Nostoc flagelliforme and induction of drnf1 transcription. ( A ) Physiologically recovered samples were treated by NaCl solutions for 27 h and then transferred to normal solutions for physiological recovery for 20 h. Physiological changes were indexed with the Fv/Fm parameter. Data are mean ± s.d. ( n = 5); ( B ) Semi-quantitative RT-PCR analysis of transcription levels of drnf1 gene under 0.4 M NaCl for different times. 16S rRNA gene was used an internal reference.

Techniques Used: Quantitative RT-PCR

10) Product Images from "Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9"

Article Title: Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E19-04-0223

CRISPR/Cas9-mediated knock-out of Syp and PSD-95 to examine the correlation of endogenous expression of pre- and postsynaptic proteins in cultured neurons. (A, C) The structure of the rat Syp locus and the nucleotide sequence of exon 4–5 (A), and the structure of rat PSD-95 locus and the nucleotide sequence of exon 3–8 (C). Intron sequences are shown in gray. The gRNA targeting sequences are underlined, and the PAM sequences are shown in bold. The predicted Cas9-gRNA cutting positions are indicated with red triangles. Arrows denote the position of the PCR primers used for genotyping. (B, D) Genomic PCR analysis of primary neuronal cultures nucleofected with the CRISPR constructs. Primary hippocampal neurons were nucleofected with CAG-EGFP and the following plasmids: CBh-Cas9 without gRNA (B and D, lane 1), a combination of CBh-Cas9/U6-gRNA (Syp#1) and CBh-Cas9/U6-gRNA (Syp#4) (B, lane 2), a combination of CBh-Cas9/U6-gRNA (Syp#2) and CBh-Cas9/U6-gRNA (Syp#3) (B, lane 3), a combination of CBh-Cas9/U6-gRNA (PSD-95#1) and CBh-Cas9/U6-gRNA (PSD-95#2) (D, lane 2), or a combination of CBh-Cas9/U6-gRNA (PSD-95#1) and CBh-Cas9/U6-gRNA (PSD-95#3) (D, lane 3). DNAs were extracted from the cultured neurons at 19 DIV and subjected to PCR analysis. Targeted genome modifications were also verified by Sanger sequencing of the PCR amplicons (see Supplemental Figures S3 and S4). (E, F) Western blot analysis of whole-cell lysates from 19 DIV rat primary hippocampal neurons nucleofected with the indicated CRISPR constructs. Immunoblots were probed with indicated antibodies, and densitometric analysis of blots was performed to determine the fold differences in endogenous expression of PSD-95 (E) or Syp (F). The graphs show quantitative densitometry and statistical analysis of the Western blot bands; n = 3 (three blot membranes from three sample sets); mean ± SEM (error bars); *p
Figure Legend Snippet: CRISPR/Cas9-mediated knock-out of Syp and PSD-95 to examine the correlation of endogenous expression of pre- and postsynaptic proteins in cultured neurons. (A, C) The structure of the rat Syp locus and the nucleotide sequence of exon 4–5 (A), and the structure of rat PSD-95 locus and the nucleotide sequence of exon 3–8 (C). Intron sequences are shown in gray. The gRNA targeting sequences are underlined, and the PAM sequences are shown in bold. The predicted Cas9-gRNA cutting positions are indicated with red triangles. Arrows denote the position of the PCR primers used for genotyping. (B, D) Genomic PCR analysis of primary neuronal cultures nucleofected with the CRISPR constructs. Primary hippocampal neurons were nucleofected with CAG-EGFP and the following plasmids: CBh-Cas9 without gRNA (B and D, lane 1), a combination of CBh-Cas9/U6-gRNA (Syp#1) and CBh-Cas9/U6-gRNA (Syp#4) (B, lane 2), a combination of CBh-Cas9/U6-gRNA (Syp#2) and CBh-Cas9/U6-gRNA (Syp#3) (B, lane 3), a combination of CBh-Cas9/U6-gRNA (PSD-95#1) and CBh-Cas9/U6-gRNA (PSD-95#2) (D, lane 2), or a combination of CBh-Cas9/U6-gRNA (PSD-95#1) and CBh-Cas9/U6-gRNA (PSD-95#3) (D, lane 3). DNAs were extracted from the cultured neurons at 19 DIV and subjected to PCR analysis. Targeted genome modifications were also verified by Sanger sequencing of the PCR amplicons (see Supplemental Figures S3 and S4). (E, F) Western blot analysis of whole-cell lysates from 19 DIV rat primary hippocampal neurons nucleofected with the indicated CRISPR constructs. Immunoblots were probed with indicated antibodies, and densitometric analysis of blots was performed to determine the fold differences in endogenous expression of PSD-95 (E) or Syp (F). The graphs show quantitative densitometry and statistical analysis of the Western blot bands; n = 3 (three blot membranes from three sample sets); mean ± SEM (error bars); *p

Techniques Used: CRISPR, Knock-Out, Expressing, Cell Culture, Sequencing, Polymerase Chain Reaction, Construct, Western Blot

Validation of CRISPR/Cas9-mediated knock-in of fluorescent genes into cultured neurons. (A, F) Structures of the rat PSD-95 (A) and Syp (F) loci and the knock-in constructs to produce EGFP and mCherry fusion proteins. The gRNA targeting sequences are underlined, and the PAM sequences are shown in red. The predicted Cas9-gRNA cutting positions are indicated with a scissor-cutting symbols. These two gRNAs for each gene were used together to increase the knock-in efficiency. Locations of genotyping primers sets to detect EGFP or mCherry knock-in alleles of PSD-95 (EGFP: rPsd-F1 and EGFP-R1; mCherry: rPSD-F1 and mCherry-R1) and Syp (EGFP: rSyp-F1 and EGFP-R1; mCherry: rSyp-F1 and mCherry-R1) are shown. (B–E) Genomic PCR analysis of nucleofected neurons. Cortical (B, D) or hippocampal (C, E) neurons were nucleofected with three plasmids: PSD-95 targeting vector (EGFP), CAG-mCherry, and CBh-Cas9 with or without gRNAs for PSD-95 (B, C), or PSD-95 targeting vector (mCherry), CAG-EGFP, and CBh-Cas9 with or without gRNAs for PSD-95 (D, E). Genomic DNA samples extracted from the cultured neurons at 21 DIV were subjected to PCR analysis. PCR primer sets were used to detect the fluorescent protein knock-in alleles, transfected targeting vectors, and endogenous rat Actb , respectively. Agarose gel electrophoresis of PCR products are shown: lane 1, DNA prepared from the primary neuron cultures nucleofected without CBh-Cas9/U6-gRNA (PSD-95); lane 2, DNA prepared from the neuron primary cultures nucleofected with CBh-Cas9/U6-gRNA (PSD-95); lane 3, wild-type rat genomic DNA; lane 4, PSD-95 targeting vector plasmid DNA. (G–J) Genomic PCR analysis of nucleofected neurons. Cortical (G, I) or hippocampal (H, J) neurons were nucleofected with three plasmids: Syp targeting vector (EGFP), CAG-mCherry, and CBh-Cas9 with or without gRNAs for Syp (G, H), or Syp targeting vector (mCherry), CAG-EGFP, and CBh-Cas9 with or without gRNAs for Syp (I, J). Genomic DNA samples extracted from the cultured neurons at 21 DIV were subjected to PCR analysis. PCR primer sets were used to detect the fluorescent protein knock-in alleles, transfected targeting vectors, and endogenous rat Actb , respectively. Agarose gel electrophoresis of PCR products are shown: lane 1, DNA prepared from the primary neuron cultures nucleofected without CBh-Cas9/U6-gRNA (Syp); lane 2, DNA prepared from the neuron primary cultures nucleofected with CBh-Cas9/U6-gRNA (Syp); lane 3, wild-type rat genomic DNA; lane 4, Syp targeting vector plasmid DNA.
Figure Legend Snippet: Validation of CRISPR/Cas9-mediated knock-in of fluorescent genes into cultured neurons. (A, F) Structures of the rat PSD-95 (A) and Syp (F) loci and the knock-in constructs to produce EGFP and mCherry fusion proteins. The gRNA targeting sequences are underlined, and the PAM sequences are shown in red. The predicted Cas9-gRNA cutting positions are indicated with a scissor-cutting symbols. These two gRNAs for each gene were used together to increase the knock-in efficiency. Locations of genotyping primers sets to detect EGFP or mCherry knock-in alleles of PSD-95 (EGFP: rPsd-F1 and EGFP-R1; mCherry: rPSD-F1 and mCherry-R1) and Syp (EGFP: rSyp-F1 and EGFP-R1; mCherry: rSyp-F1 and mCherry-R1) are shown. (B–E) Genomic PCR analysis of nucleofected neurons. Cortical (B, D) or hippocampal (C, E) neurons were nucleofected with three plasmids: PSD-95 targeting vector (EGFP), CAG-mCherry, and CBh-Cas9 with or without gRNAs for PSD-95 (B, C), or PSD-95 targeting vector (mCherry), CAG-EGFP, and CBh-Cas9 with or without gRNAs for PSD-95 (D, E). Genomic DNA samples extracted from the cultured neurons at 21 DIV were subjected to PCR analysis. PCR primer sets were used to detect the fluorescent protein knock-in alleles, transfected targeting vectors, and endogenous rat Actb , respectively. Agarose gel electrophoresis of PCR products are shown: lane 1, DNA prepared from the primary neuron cultures nucleofected without CBh-Cas9/U6-gRNA (PSD-95); lane 2, DNA prepared from the neuron primary cultures nucleofected with CBh-Cas9/U6-gRNA (PSD-95); lane 3, wild-type rat genomic DNA; lane 4, PSD-95 targeting vector plasmid DNA. (G–J) Genomic PCR analysis of nucleofected neurons. Cortical (G, I) or hippocampal (H, J) neurons were nucleofected with three plasmids: Syp targeting vector (EGFP), CAG-mCherry, and CBh-Cas9 with or without gRNAs for Syp (G, H), or Syp targeting vector (mCherry), CAG-EGFP, and CBh-Cas9 with or without gRNAs for Syp (I, J). Genomic DNA samples extracted from the cultured neurons at 21 DIV were subjected to PCR analysis. PCR primer sets were used to detect the fluorescent protein knock-in alleles, transfected targeting vectors, and endogenous rat Actb , respectively. Agarose gel electrophoresis of PCR products are shown: lane 1, DNA prepared from the primary neuron cultures nucleofected without CBh-Cas9/U6-gRNA (Syp); lane 2, DNA prepared from the neuron primary cultures nucleofected with CBh-Cas9/U6-gRNA (Syp); lane 3, wild-type rat genomic DNA; lane 4, Syp targeting vector plasmid DNA.

Techniques Used: CRISPR, Knock-In, Cell Culture, Construct, Polymerase Chain Reaction, Plasmid Preparation, Transfection, Agarose Gel Electrophoresis

11) Product Images from "Psy2 Targets the PP4 Family Phosphatase Pph3 To Dephosphorylate Mth1 and Repress Glucose Transporter Gene Expression"

Article Title: Psy2 Targets the PP4 Family Phosphatase Pph3 To Dephosphorylate Mth1 and Repress Glucose Transporter Gene Expression

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00279-13

Mth1-4SA mutant counteracts the effect of psy2 deletion on HXT3 expression. (A) Mth1-4SA mutant restores the induction of HXT3 gene in psy2Δ cells to wild-type level, as determined by quantitative RT-PCR. (B) Mth1-4SA mutant rescues the delayed repression of HXT3 gene in psy2Δ cells in the absence of glucose, as determined by quantitative RT-PCR. (C) Mth1-4SA mutation does not lead to significant changes in the Mth1 degradation pattern. An anti-PSTAIRE antibody immunoblot was used as a loading control.
Figure Legend Snippet: Mth1-4SA mutant counteracts the effect of psy2 deletion on HXT3 expression. (A) Mth1-4SA mutant restores the induction of HXT3 gene in psy2Δ cells to wild-type level, as determined by quantitative RT-PCR. (B) Mth1-4SA mutant rescues the delayed repression of HXT3 gene in psy2Δ cells in the absence of glucose, as determined by quantitative RT-PCR. (C) Mth1-4SA mutation does not lead to significant changes in the Mth1 degradation pattern. An anti-PSTAIRE antibody immunoblot was used as a loading control.

Techniques Used: Mutagenesis, Expressing, Quantitative RT-PCR

Psy2 promotes HXT gene repression in the absence of glucose. (A) The phosphorylation state of Rgt1 was analyzed by immunoblotting using wild-type (WT) and psy2Δ mutant cells grown in Gal-containing medium and shifted to Glc-containing medium. The position of unphosphorylated Rgt1 is indicated by a line, whereas the lines with asterisks denote the most highly phosphorylated species of Rgt1. The membrane was stained for protein prior to development for the loading control. (B) Similar analysis was performed with WT and psy2Δ mutant cells shifted from Glc- to Gal-containing medium for the time points indicated. An immunoblot with anti-PSTAIRE antibody was used for the loading control. (C) Quantitative RT-PCR analysis of HXT3 RNA with samples collected as described in panel B at the indicated time points. (D) Chromatin immunoprecipitation assay was performed using wild-type (WT) and psy2Δ mutant cells treated as in panel B, after cross-linking and immunoprecipitation of the Rgt1-HA-DNA complex using anti-HA monoclonal antibodies. The qPCR data represent the average of HXT3 RNA samples analyzed in triplicate and normalized to ACT1 RNA. HXT3 RNA is plotted relative to the wild-type time zero sample.
Figure Legend Snippet: Psy2 promotes HXT gene repression in the absence of glucose. (A) The phosphorylation state of Rgt1 was analyzed by immunoblotting using wild-type (WT) and psy2Δ mutant cells grown in Gal-containing medium and shifted to Glc-containing medium. The position of unphosphorylated Rgt1 is indicated by a line, whereas the lines with asterisks denote the most highly phosphorylated species of Rgt1. The membrane was stained for protein prior to development for the loading control. (B) Similar analysis was performed with WT and psy2Δ mutant cells shifted from Glc- to Gal-containing medium for the time points indicated. An immunoblot with anti-PSTAIRE antibody was used for the loading control. (C) Quantitative RT-PCR analysis of HXT3 RNA with samples collected as described in panel B at the indicated time points. (D) Chromatin immunoprecipitation assay was performed using wild-type (WT) and psy2Δ mutant cells treated as in panel B, after cross-linking and immunoprecipitation of the Rgt1-HA-DNA complex using anti-HA monoclonal antibodies. The qPCR data represent the average of HXT3 RNA samples analyzed in triplicate and normalized to ACT1 RNA. HXT3 RNA is plotted relative to the wild-type time zero sample.

Techniques Used: Mutagenesis, Gas Chromatography, Staining, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

12) Product Images from "Optimization of soybean (Glycine max (L.) Merrill) in planta ovary transformation using a linear minimal gus gene cassette *"

Article Title: Optimization of soybean (Glycine max (L.) Merrill) in planta ovary transformation using a linear minimal gus gene cassette *

Journal: Journal of Zhejiang University. Science. B

doi: 10.1631/jzus.B0920204

RT-PCR analysis of gus expression in T 1 plants
Figure Legend Snippet: RT-PCR analysis of gus expression in T 1 plants

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

13) Product Images from "BCL2 Inhibitor (ABT-737): A Restorer of Prednisolone Sensitivity in Early T-Cell Precursor-Acute Lymphoblastic Leukemia with High MEF2C Expression?"

Article Title: BCL2 Inhibitor (ABT-737): A Restorer of Prednisolone Sensitivity in Early T-Cell Precursor-Acute Lymphoblastic Leukemia with High MEF2C Expression?

Journal: PLoS ONE

doi: 10.1371/journal.pone.0132926

MEF2C expression and PSL sensitivity of primary leukemic blast cells in four T-ALL patients (two ETP-ALL and two non-ETP ALL). (a) MEF2C expression levels determined by real-time quantitative-PCR. GAPDH was used as an internal control. (b) Viability of cells was counted after treatment with PSL (200 μM) and/or ABT-737 (10 nM). Annexin (-)/PI(-) cells were defined as viable cells.
Figure Legend Snippet: MEF2C expression and PSL sensitivity of primary leukemic blast cells in four T-ALL patients (two ETP-ALL and two non-ETP ALL). (a) MEF2C expression levels determined by real-time quantitative-PCR. GAPDH was used as an internal control. (b) Viability of cells was counted after treatment with PSL (200 μM) and/or ABT-737 (10 nM). Annexin (-)/PI(-) cells were defined as viable cells.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Expression levels of MEF2C and FLT3 in ETP-ALL vs. typical T-ALL blast cells. Comparison of the expression levels of MEF2C and FLT3 in ETP-ALL vs. typical T-ALL blast cells determined by real-time quantitative-PCR analysis.
Figure Legend Snippet: Expression levels of MEF2C and FLT3 in ETP-ALL vs. typical T-ALL blast cells. Comparison of the expression levels of MEF2C and FLT3 in ETP-ALL vs. typical T-ALL blast cells determined by real-time quantitative-PCR analysis.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Comparison of LOUCY and Jurkat leukemia cells. (a) Expression of MEF2C and β-actin by western blot analyses. (b), (c) Cell growth inhibition of leukemia cells assessed by WST assay with serial concentrations of prednisolone (PSL) or ABT-737. (d) q-PCR analysis of BCL2 of two cell lines 12 hours after treatment of ABT-737 with the concentration of 10 or 100 nM. The bar indicates the mean±SE of two independent experiments in triplicate (e) Cell growth inhibition with serial concentrations of prednisolone (PSL) combined with ABT-737 (10 nM). (f) Calculation of the IC50 of PSL, following treatment of leukemic cells with either PSL, or a combination of PSL and ABT-737. Calculated combination Index of less than 1.0 was considered as a synergistic effect. (g), (h) Apoptosis study. Annexin V-positive cells were counted as apoptotic. Flow cytometry data in (g) and the mean±SE of three independent experiments in (h).
Figure Legend Snippet: Comparison of LOUCY and Jurkat leukemia cells. (a) Expression of MEF2C and β-actin by western blot analyses. (b), (c) Cell growth inhibition of leukemia cells assessed by WST assay with serial concentrations of prednisolone (PSL) or ABT-737. (d) q-PCR analysis of BCL2 of two cell lines 12 hours after treatment of ABT-737 with the concentration of 10 or 100 nM. The bar indicates the mean±SE of two independent experiments in triplicate (e) Cell growth inhibition with serial concentrations of prednisolone (PSL) combined with ABT-737 (10 nM). (f) Calculation of the IC50 of PSL, following treatment of leukemic cells with either PSL, or a combination of PSL and ABT-737. Calculated combination Index of less than 1.0 was considered as a synergistic effect. (g), (h) Apoptosis study. Annexin V-positive cells were counted as apoptotic. Flow cytometry data in (g) and the mean±SE of three independent experiments in (h).

Techniques Used: Expressing, Western Blot, Inhibition, WST Assay, Polymerase Chain Reaction, Concentration Assay, Flow Cytometry, Cytometry

14) Product Images from "Detection of PCV2 DNA by SYBR Green I-based quantitative PCR"

Article Title: Detection of PCV2 DNA by SYBR Green I-based quantitative PCR

Journal:

doi: 10.1631/jzus.2007.B0162

Regression lines between the C T values and the input concentrations of PCV2 plasmid DNA in real-time PCR detected by using SYBR Green I
Figure Legend Snippet: Regression lines between the C T values and the input concentrations of PCV2 plasmid DNA in real-time PCR detected by using SYBR Green I

Techniques Used: Plasmid Preparation, Real-time Polymerase Chain Reaction, SYBR Green Assay

15) Product Images from "Chromosomal Insertions in the Lactobacillus caseiupp Gene That Are Useful for Vaccine Expression"

Article Title: Chromosomal Insertions in the Lactobacillus caseiupp Gene That Are Useful for Vaccine Expression

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00175-14

Schematic diagram of vector construction. 1, PCR products of upstream homologous arm 2A with AscI and BglII, cloned into the pMD18-T vector and named pMD18-T-2A. pMD18-T-2A was digested with AscI and BglII endonucleases and inserted into the pGBHCupp
Figure Legend Snippet: Schematic diagram of vector construction. 1, PCR products of upstream homologous arm 2A with AscI and BglII, cloned into the pMD18-T vector and named pMD18-T-2A. pMD18-T-2A was digested with AscI and BglII endonucleases and inserted into the pGBHCupp

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Clone Assay

16) Product Images from "Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides"

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides

Journal: Microbial Cell Factories

doi: 10.1186/s12934-019-1110-4

Structure of plasmids pMAT1552 and pMAT1552-GLELO are shown. The D6E ( GLELO) gene was isolated by PCR amplification with appropriate primers. The PCR fragment was ligated into XhoI restriction site to generate plasmid as pMAT1552-GLELO
Figure Legend Snippet: Structure of plasmids pMAT1552 and pMAT1552-GLELO are shown. The D6E ( GLELO) gene was isolated by PCR amplification with appropriate primers. The PCR fragment was ligated into XhoI restriction site to generate plasmid as pMAT1552-GLELO

Techniques Used: Isolation, Polymerase Chain Reaction, Amplification, Plasmid Preparation

17) Product Images from "Identification of a Novel Nurr1-Interacting Protein"

Article Title: Identification of a Novel Nurr1-Interacting Protein

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3021-08.2008

Alternative splicing of NuIP gene and the tissue distribution of different transcript isoforms. A , Schematic representation of the putative alternatively spliced isoforms of NuIP gene. B , RT-PCR, using primers specific for the alternatively spliced NuIP transcripts, Nurr1, and GAPDH, were performed on mRNAs extracted from different mouse tissues. Nurr1 and full-length NuIP transcripts were consistently coexpressed in the various tissues examined.
Figure Legend Snippet: Alternative splicing of NuIP gene and the tissue distribution of different transcript isoforms. A , Schematic representation of the putative alternatively spliced isoforms of NuIP gene. B , RT-PCR, using primers specific for the alternatively spliced NuIP transcripts, Nurr1, and GAPDH, were performed on mRNAs extracted from different mouse tissues. Nurr1 and full-length NuIP transcripts were consistently coexpressed in the various tissues examined.

Techniques Used: Reverse Transcription Polymerase Chain Reaction

Effects of NuIP knockdown in an engineered MN9D cell line. A , Domain structure of NuIP and sites targeted by inducibly expressed siRNA in corresponding mRNA. RUN domain, aa 44–189; TBC domain, 881–1053. siRNA generated from RNAi#1 vector targets the junction of exons 12 and 13, and siRNA from RNAi#2 targets exon 8. B , Inducible knockdown of NuIP protein. Stable MN9D cells expressing tetracycline repressor were transfected with pSUPERIOR constructs with or without siDNA inserts. Twenty-four hours after transfection, 2 μg/ml of DOX was added to induce siRNA expression. The cells were harvested 72 h after induction. Cell lysates were analyzed for NuIP, DAT, TH, and α-tubulin by Western blot. C , Inducible knockdown of NuIP mRNA. Stable MN9D cells expressing tetracycline repressor were transfected with pSUPERIOR constructs with or without siDNA inserts. Twenty four hours after transfection, 2 μg/ml of DOX was added to induce siRNA expression, and RNA was isolated 72 h after induction. NuIP transcript levels were quantified with qRT-PCR and normalized to 18S ribosomal RNA; averages of triplicate quantification are shown. Error bars indicate SD. D , MN9D cell numbers after inducible RNAi knockdown of NuIP mRNA. Stable MN9D cells expressing tetracycline repressor were treated the same way as described above. The results are means from seven independent experiments; error bars indicate SD. E , Downregulation of the DAT in stably transfected MN9D cells in which NuIP was knocked down. Cells grown as above were harvested 72 h after RNAi induction, protein lysates prepared, samples analyzed via Western blots, lanes scanned, and the intensity of DAT and TH expression determined. A significant difference was observed between mock and RNAi for DAT ( p = 0.006). The results shown represent the means of 2–4 independent experiments. Error bars indicate SD. * p
Figure Legend Snippet: Effects of NuIP knockdown in an engineered MN9D cell line. A , Domain structure of NuIP and sites targeted by inducibly expressed siRNA in corresponding mRNA. RUN domain, aa 44–189; TBC domain, 881–1053. siRNA generated from RNAi#1 vector targets the junction of exons 12 and 13, and siRNA from RNAi#2 targets exon 8. B , Inducible knockdown of NuIP protein. Stable MN9D cells expressing tetracycline repressor were transfected with pSUPERIOR constructs with or without siDNA inserts. Twenty-four hours after transfection, 2 μg/ml of DOX was added to induce siRNA expression. The cells were harvested 72 h after induction. Cell lysates were analyzed for NuIP, DAT, TH, and α-tubulin by Western blot. C , Inducible knockdown of NuIP mRNA. Stable MN9D cells expressing tetracycline repressor were transfected with pSUPERIOR constructs with or without siDNA inserts. Twenty four hours after transfection, 2 μg/ml of DOX was added to induce siRNA expression, and RNA was isolated 72 h after induction. NuIP transcript levels were quantified with qRT-PCR and normalized to 18S ribosomal RNA; averages of triplicate quantification are shown. Error bars indicate SD. D , MN9D cell numbers after inducible RNAi knockdown of NuIP mRNA. Stable MN9D cells expressing tetracycline repressor were treated the same way as described above. The results are means from seven independent experiments; error bars indicate SD. E , Downregulation of the DAT in stably transfected MN9D cells in which NuIP was knocked down. Cells grown as above were harvested 72 h after RNAi induction, protein lysates prepared, samples analyzed via Western blots, lanes scanned, and the intensity of DAT and TH expression determined. A significant difference was observed between mock and RNAi for DAT ( p = 0.006). The results shown represent the means of 2–4 independent experiments. Error bars indicate SD. * p

Techniques Used: Generated, Plasmid Preparation, Expressing, Transfection, Construct, Western Blot, Isolation, Quantitative RT-PCR, Stable Transfection

18) Product Images from "Regulation of the PI3K/Akt pathway during decidualization of endometrial stromal cells"

Article Title: Regulation of the PI3K/Akt pathway during decidualization of endometrial stromal cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0177387

Effect of forced expression of Akt isoforms (CA-Akt) on PRL and IGFBP1 expression. mRNA expression of either PRL (A) IGFBP1 (B) were quantified following three days treatments. HIESC cells transfected with either Akt1, Akt2 or Akt3 Tet-On vectors were subjected to decidualization using cAMP (0.5 mM) and MPA (10μM). They were then either concomitantly treated with doxycycline (1μg/mL) in order to induce the expression of the constitutive Akt isoform construct (cAMP+MPA+Doxycycline) or cells were allowed to decidualize for 24h before doxycycline was added (cAMP+MPA+Doxycycline 24H). Cells were lysed after three days and qRT-PCR analyses were performed to quantify PRL or IGBP1 expression. β-actin mRNA expression was used as control for qPCR results. (C) Akt 1, Akt2 and Akt3 expression was quantified following three days treatments. HIESC cells transfected with either Akt1, Akt2 or Akt3 Tet-On vectors were subjected to decidualization using cAMP (0.5 mM) and MPA (10μM). They were then either concomitantly treated with doxycycline (1μg/mL) in order to induce the expression of the constitutive Akt isoform construct (cAMP+MPA+Doxycycline). Cells were lysed after three days and qRT-PCR analyses were performed to quantify Akt1, Akt2 or Akt3 expression. β-actin mRNA expression was used as control for qPCR results. Data are means ± SEM three independent experiments. Different letters represent significantly different means (p
Figure Legend Snippet: Effect of forced expression of Akt isoforms (CA-Akt) on PRL and IGFBP1 expression. mRNA expression of either PRL (A) IGFBP1 (B) were quantified following three days treatments. HIESC cells transfected with either Akt1, Akt2 or Akt3 Tet-On vectors were subjected to decidualization using cAMP (0.5 mM) and MPA (10μM). They were then either concomitantly treated with doxycycline (1μg/mL) in order to induce the expression of the constitutive Akt isoform construct (cAMP+MPA+Doxycycline) or cells were allowed to decidualize for 24h before doxycycline was added (cAMP+MPA+Doxycycline 24H). Cells were lysed after three days and qRT-PCR analyses were performed to quantify PRL or IGBP1 expression. β-actin mRNA expression was used as control for qPCR results. (C) Akt 1, Akt2 and Akt3 expression was quantified following three days treatments. HIESC cells transfected with either Akt1, Akt2 or Akt3 Tet-On vectors were subjected to decidualization using cAMP (0.5 mM) and MPA (10μM). They were then either concomitantly treated with doxycycline (1μg/mL) in order to induce the expression of the constitutive Akt isoform construct (cAMP+MPA+Doxycycline). Cells were lysed after three days and qRT-PCR analyses were performed to quantify Akt1, Akt2 or Akt3 expression. β-actin mRNA expression was used as control for qPCR results. Data are means ± SEM three independent experiments. Different letters represent significantly different means (p

Techniques Used: Expressing, Transfection, Construct, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

19) Product Images from "Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414"

Article Title: Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414

Journal: Applied and Environmental Microbiology

doi:

Restriction map and sequencing strategy of the genomic DNA for T. reesei egl3 (A) and the complete nucleotide sequence of the gene and deduced amino acid sequence of the EG III protein (B). (A) The Hin dIII fragment of the egl3 genomic clones is shown as a bar, and the egl3 structural gene region is shown as a filled box. The orientations and lengths of coverage of sequencing primers are shown as horizontal arrows. (B) Intron sequences are in lowercase type. The standard one-letter amino acid code is used. The presumed signal sequence is indicated by the dotted underline. The internal amino acid sequences determined for the lysylendopeptidase-digested peptides of the purified T. reesei EG III are underlined. The amino acid sequences for the design of PCR primers are double underlined.
Figure Legend Snippet: Restriction map and sequencing strategy of the genomic DNA for T. reesei egl3 (A) and the complete nucleotide sequence of the gene and deduced amino acid sequence of the EG III protein (B). (A) The Hin dIII fragment of the egl3 genomic clones is shown as a bar, and the egl3 structural gene region is shown as a filled box. The orientations and lengths of coverage of sequencing primers are shown as horizontal arrows. (B) Intron sequences are in lowercase type. The standard one-letter amino acid code is used. The presumed signal sequence is indicated by the dotted underline. The internal amino acid sequences determined for the lysylendopeptidase-digested peptides of the purified T. reesei EG III are underlined. The amino acid sequences for the design of PCR primers are double underlined.

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

20) Product Images from "Activation of Cell Cycle Arrest and Apoptosis by the Proto-Oncogene Pim-2"

Article Title: Activation of Cell Cycle Arrest and Apoptosis by the Proto-Oncogene Pim-2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034736

Up-regulation of E2F-1 and p73 expression in HeLa cells over-expressing the 34 kDa form of PIM-2. ( A ) RT-PCR analysis of E2F-1 transcripts in Pim2 over-expressing cells (Pim-2) and in control cells (HA). Actin specific primers were used as reference for equal loading. ( B ) Western blot analysis of the E2F-1 protein in nuclear extracts (40 µg) from cells over-expressing Pim-2 (Pim-2) and control cells with empty HA vector (HA). Blot was stripped and reprobed with antibodies specific to RCC1 as reference for equal loading. The average relative level of E2F-1 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is shown in the right panel. Comparison was based on densitometric analysis of E2F-1 signals normalized according to the RCC1 signal (average of three independent experiments). Average level of E2F-1 in control cells was determined as 1. Asterisk represents statistically significant differences (p
Figure Legend Snippet: Up-regulation of E2F-1 and p73 expression in HeLa cells over-expressing the 34 kDa form of PIM-2. ( A ) RT-PCR analysis of E2F-1 transcripts in Pim2 over-expressing cells (Pim-2) and in control cells (HA). Actin specific primers were used as reference for equal loading. ( B ) Western blot analysis of the E2F-1 protein in nuclear extracts (40 µg) from cells over-expressing Pim-2 (Pim-2) and control cells with empty HA vector (HA). Blot was stripped and reprobed with antibodies specific to RCC1 as reference for equal loading. The average relative level of E2F-1 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is shown in the right panel. Comparison was based on densitometric analysis of E2F-1 signals normalized according to the RCC1 signal (average of three independent experiments). Average level of E2F-1 in control cells was determined as 1. Asterisk represents statistically significant differences (p

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation

p57 expression and T14/Y15 phosphorylation on CDK2 in HeLa cells over-expressing the 34 kDa PIM-2 isoform. ( A ) RT-PCR analysis of p57 transcripts in control cells (HA control) and in Pim2 over-expressing cells (HA Pim-2). Actin specific primers were used as reference for equal loading. Western blot analysis was used to evaluate the p57 protein in nuclear extracts (40 µg) from HA control cells and from cells over-expressing Pim-2 (HA Pim-2). Anti-RCC1 antibodies were used as reference for equal loading. The average relative level of p57 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is depicted in the right panel. Comparison was based on densitometric analysis of p57 signals normalized according to the RCC1 signal (average of three independent experiments). Average level of p57 in control cells was determined as 1. Differences were just above the statistically significance of p
Figure Legend Snippet: p57 expression and T14/Y15 phosphorylation on CDK2 in HeLa cells over-expressing the 34 kDa PIM-2 isoform. ( A ) RT-PCR analysis of p57 transcripts in control cells (HA control) and in Pim2 over-expressing cells (HA Pim-2). Actin specific primers were used as reference for equal loading. Western blot analysis was used to evaluate the p57 protein in nuclear extracts (40 µg) from HA control cells and from cells over-expressing Pim-2 (HA Pim-2). Anti-RCC1 antibodies were used as reference for equal loading. The average relative level of p57 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is depicted in the right panel. Comparison was based on densitometric analysis of p57 signals normalized according to the RCC1 signal (average of three independent experiments). Average level of p57 in control cells was determined as 1. Differences were just above the statistically significance of p

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

21) Product Images from "Efficient Generation of diRNAs Requires Components in the Posttranscriptional Gene Silencing Pathway"

Article Title: Efficient Generation of diRNAs Requires Components in the Posttranscriptional Gene Silencing Pathway

Journal: Scientific Reports

doi: 10.1038/s41598-017-00374-7

diRNAs are not detected at endogenous repeat regions. ( A – C ) The repeat regions and the targeted T1 sequences are shown for the ( A ) At1g31290 repeat1, ( B ) At1g31290 repeat2, and ( C ) At5g54700, respectively. The top gray and black horizontal bars represent the repeats, and the CRISPR/Cas9 target sites are indicated by arrows. The sequence of the repeats is below the gray/black schematic. Below the reference repeat sequence are the sequencing results from independent T1 plants. See also Supplemental Figs 4 and 5 . ( D – F ) Northern blot analysis for small RNAs in the indicated Col0 control, T1, T2 with CRISPR/Cas9 transgene (T2 w) or without CRISPR/Cas9 transgene (T2 wo). ( D ) At1g31290 repeat1, ( E ) At1g31290 repeat2, and ( F ) At5g54700, respectively. miR167 was probed as control. Northern blotting image was cropped nearby signals. ( G ) qRT-PCR detection of antisense transcripts in At1g31290 repeats targeted by CRISPR/Cas9. At1g31290-as indicates At1g31290 antisense transcript. IGN33 was analyzed as control. Black bar; Col0 with RT, gray bar; At1g31290 targeting CRIPR/Cas9 with RT, white bar; without RT control. N = 3. N.D.; not detected.
Figure Legend Snippet: diRNAs are not detected at endogenous repeat regions. ( A – C ) The repeat regions and the targeted T1 sequences are shown for the ( A ) At1g31290 repeat1, ( B ) At1g31290 repeat2, and ( C ) At5g54700, respectively. The top gray and black horizontal bars represent the repeats, and the CRISPR/Cas9 target sites are indicated by arrows. The sequence of the repeats is below the gray/black schematic. Below the reference repeat sequence are the sequencing results from independent T1 plants. See also Supplemental Figs 4 and 5 . ( D – F ) Northern blot analysis for small RNAs in the indicated Col0 control, T1, T2 with CRISPR/Cas9 transgene (T2 w) or without CRISPR/Cas9 transgene (T2 wo). ( D ) At1g31290 repeat1, ( E ) At1g31290 repeat2, and ( F ) At5g54700, respectively. miR167 was probed as control. Northern blotting image was cropped nearby signals. ( G ) qRT-PCR detection of antisense transcripts in At1g31290 repeats targeted by CRISPR/Cas9. At1g31290-as indicates At1g31290 antisense transcript. IGN33 was analyzed as control. Black bar; Col0 with RT, gray bar; At1g31290 targeting CRIPR/Cas9 with RT, white bar; without RT control. N = 3. N.D.; not detected.

Techniques Used: CRISPR, Sequencing, Northern Blot, Quantitative RT-PCR

Efficient diRNA production requires active transcription. ( A ) Representative GUS staining images of GU-US reporter system in T1. ( B ) Representative GUS staining images of functional GUS targeted by CRIPSR/Cas9 in T1. ( C ) Detection of GU-US transgene repair by PCR. The longer fragment indicates the parental (unrepaired) GU-US, and the shorter fragment indicates the functional GUS generated by HR. The PCR product size of GU-US and GUS are 1446 bp and 888 bp, respectively. Electrophoresis gel image was cropped nearby signals. ( D ) Detection of small RNAs by Northern blot. The U of GUS gene was as a probe. U6 was probed as control. Northern blotting image was cropped nearby signals. ( E ) The distribution of small RNAs around the GU-US transgene, under the indicated conditions. Two independent T1 transgenic lines for each construct were analyzed. ( F ) The distribution of small RNA for GUS transgene, under the indicated conditions. Two independent T1 transgenic lines for each construct were analyzed. ( E , F ) The y axis represents the number of small RNA reads within 100 bp sliding windows with a step size of 1 bp, numbers in (+) and (−) values represent the reads of 21 nt in length small RNAs derived from sense and antisense strands, respectively. The vertical arrows indicate CRISPR/Cas9 target sites. ( A – F ) 35S promoter and CRIPSR/Cas9 genotype were indicated as with (+) or without (−).
Figure Legend Snippet: Efficient diRNA production requires active transcription. ( A ) Representative GUS staining images of GU-US reporter system in T1. ( B ) Representative GUS staining images of functional GUS targeted by CRIPSR/Cas9 in T1. ( C ) Detection of GU-US transgene repair by PCR. The longer fragment indicates the parental (unrepaired) GU-US, and the shorter fragment indicates the functional GUS generated by HR. The PCR product size of GU-US and GUS are 1446 bp and 888 bp, respectively. Electrophoresis gel image was cropped nearby signals. ( D ) Detection of small RNAs by Northern blot. The U of GUS gene was as a probe. U6 was probed as control. Northern blotting image was cropped nearby signals. ( E ) The distribution of small RNAs around the GU-US transgene, under the indicated conditions. Two independent T1 transgenic lines for each construct were analyzed. ( F ) The distribution of small RNA for GUS transgene, under the indicated conditions. Two independent T1 transgenic lines for each construct were analyzed. ( E , F ) The y axis represents the number of small RNA reads within 100 bp sliding windows with a step size of 1 bp, numbers in (+) and (−) values represent the reads of 21 nt in length small RNAs derived from sense and antisense strands, respectively. The vertical arrows indicate CRISPR/Cas9 target sites. ( A – F ) 35S promoter and CRIPSR/Cas9 genotype were indicated as with (+) or without (−).

Techniques Used: Staining, Functional Assay, Polymerase Chain Reaction, Generated, Electrophoresis, Northern Blot, Transgenic Assay, Construct, Derivative Assay, CRISPR

Genetic requirements for efficient diRNA generation. ( A ) GU-US transgene expression in PTGS mutants. Relative expression was determined by qRT-PCR in PTGS mutants. Error bars indicate standard error of 5 independent T1 transgenic lines. ( B ) Relative HR rate determined by qPCR. Error bars indicate standard error of 5 independent T1 transgenic lines. ( C ) Detection of small RNAs. The U part of GUS gene was used as a probe. miR167 was probed as a loading control. Northern blotting image was cropped nearby signals.
Figure Legend Snippet: Genetic requirements for efficient diRNA generation. ( A ) GU-US transgene expression in PTGS mutants. Relative expression was determined by qRT-PCR in PTGS mutants. Error bars indicate standard error of 5 independent T1 transgenic lines. ( B ) Relative HR rate determined by qPCR. Error bars indicate standard error of 5 independent T1 transgenic lines. ( C ) Detection of small RNAs. The U part of GUS gene was used as a probe. miR167 was probed as a loading control. Northern blotting image was cropped nearby signals.

Techniques Used: Expressing, Quantitative RT-PCR, Transgenic Assay, Real-time Polymerase Chain Reaction, Northern Blot

diRNAs are not detected at endogenous genes in Arabidopsis and rice. ( A ) Three independent CRISPR/Cas9 constructs target to AtBRI1 . The top panel is a schematic representation of AtBRI1 gene and three CRISPR/Cas9 gRNA target sites. Northern blot analysis was performed with LNA probes (see Supplemental Table 1 ) in AtBRI1 gene targeted CRISPR/Cas9 T1 transgenic lines. An miR167 probe was used as a control. ( B ) Northern blot analysis of small RNAs for additional endogenous genes ( GAI , AtGL2 , At5g36250, At2g36490 and At5g04560) targeted by CRISPR/Cas9. The Col0 accession and T1 transgenic plants were analyzed. ( C ) Detection of antisense transcripts at GAI by qRT-PCR, after targeting by CRIPR/Cas9. The same transgenic plants were used as in Fig. 2B. GAI -as indicates GAI antisense transcript. IGN33 was analyzed as a control. Black bar; Col0 with RT, gray bar; GAI targeting CRIPR/Cas9 with RT, white bar; without RT control. N = 3. N.D.; not detected. ( D , E ) Detection of small RNAs in endogenous genes targeted by CRISPR/Cas9 ( D ) and TALEN ( E ) in T0 transgenic rice. Horizontal line indicates TALEN target site and probe. U6 and miR159 were probed as controls. ( A , B , D , E ) Northern blotting image was cropped nearby signals.
Figure Legend Snippet: diRNAs are not detected at endogenous genes in Arabidopsis and rice. ( A ) Three independent CRISPR/Cas9 constructs target to AtBRI1 . The top panel is a schematic representation of AtBRI1 gene and three CRISPR/Cas9 gRNA target sites. Northern blot analysis was performed with LNA probes (see Supplemental Table 1 ) in AtBRI1 gene targeted CRISPR/Cas9 T1 transgenic lines. An miR167 probe was used as a control. ( B ) Northern blot analysis of small RNAs for additional endogenous genes ( GAI , AtGL2 , At5g36250, At2g36490 and At5g04560) targeted by CRISPR/Cas9. The Col0 accession and T1 transgenic plants were analyzed. ( C ) Detection of antisense transcripts at GAI by qRT-PCR, after targeting by CRIPR/Cas9. The same transgenic plants were used as in Fig. 2B. GAI -as indicates GAI antisense transcript. IGN33 was analyzed as a control. Black bar; Col0 with RT, gray bar; GAI targeting CRIPR/Cas9 with RT, white bar; without RT control. N = 3. N.D.; not detected. ( D , E ) Detection of small RNAs in endogenous genes targeted by CRISPR/Cas9 ( D ) and TALEN ( E ) in T0 transgenic rice. Horizontal line indicates TALEN target site and probe. U6 and miR159 were probed as controls. ( A , B , D , E ) Northern blotting image was cropped nearby signals.

Techniques Used: CRISPR, Construct, Northern Blot, Transgenic Assay, Quantitative RT-PCR

22) Product Images from "Zinc-Finger Antiviral Protein Inhibits XMRV Infection"

Article Title: Zinc-Finger Antiviral Protein Inhibits XMRV Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039159

Downregulation of RIG-I does not affect ZAP's antiviral activity against XMRV-luc. (A) Control shRNA and shRNAs against RIG-I (Ri446 and Ri583) were stably expressed in 293TRex-hZAP-v2 cells. RIG-I mRNA levels were measured by real-time PCR and normalized to that of GAPDH. Data presented are means ± SE of three parallel experiments. (B) Cells were transfected with pGl3-IFNβ-luc and pRL-TK. At 48 h posttransfection, cells were transfected with poly (I:C). Luciferase activity was assayed 12 h later. (C) Cells were infected with XMRV-luc, mock treated or treated with tetracycline for 48 h to induce ZAP expression, and luciferase activities were measured. Fold inhibition was calculated as the luciferase activity in mock treated cells divided by that in tetracycline treated cells. Data presented are means ± SE of three parallel experiments.
Figure Legend Snippet: Downregulation of RIG-I does not affect ZAP's antiviral activity against XMRV-luc. (A) Control shRNA and shRNAs against RIG-I (Ri446 and Ri583) were stably expressed in 293TRex-hZAP-v2 cells. RIG-I mRNA levels were measured by real-time PCR and normalized to that of GAPDH. Data presented are means ± SE of three parallel experiments. (B) Cells were transfected with pGl3-IFNβ-luc and pRL-TK. At 48 h posttransfection, cells were transfected with poly (I:C). Luciferase activity was assayed 12 h later. (C) Cells were infected with XMRV-luc, mock treated or treated with tetracycline for 48 h to induce ZAP expression, and luciferase activities were measured. Fold inhibition was calculated as the luciferase activity in mock treated cells divided by that in tetracycline treated cells. Data presented are means ± SE of three parallel experiments.

Techniques Used: Activity Assay, shRNA, Stable Transfection, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Infection, Expressing, Inhibition

Downregulation of endogenous hZAP enhances XMRV-luc expression. HOS cells were transfected with control siRNA (Ctrl) or siRNAs directed against hZAP (ZAPi-1 and ZAPi-2), followed by infection with XMRV-luc for 5 h. At 48 h postinfection, cells were lysed. (A) Endogenous hZAP mRNA levels were measured by real-time PCR. (B) Luciferase activity was measured and presented as relative light units (RLU). Data presented are means ± SD of three measurements.
Figure Legend Snippet: Downregulation of endogenous hZAP enhances XMRV-luc expression. HOS cells were transfected with control siRNA (Ctrl) or siRNAs directed against hZAP (ZAPi-1 and ZAPi-2), followed by infection with XMRV-luc for 5 h. At 48 h postinfection, cells were lysed. (A) Endogenous hZAP mRNA levels were measured by real-time PCR. (B) Luciferase activity was measured and presented as relative light units (RLU). Data presented are means ± SD of three measurements.

Techniques Used: Expressing, Transfection, Infection, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

23) Product Images from "Tomato Yellow Leaf Curl Virus V2 Interacts with Host Histone Deacetylase 6 To Suppress Methylation-Mediated Transcriptional Gene Silencing in Plants"

Article Title: Tomato Yellow Leaf Curl Virus V2 Interacts with Host Histone Deacetylase 6 To Suppress Methylation-Mediated Transcriptional Gene Silencing in Plants

Journal: Journal of Virology

doi: 10.1128/JVI.00036-18

NbHDA6 positively regulated the cytosine methylation of TYLCV. (A) Analysis of DNA methylation of the TYLCV genome by methylation-sensitive PCR. Genomic DNA was digested with HpaII, MspI, AluI, or McrBC and then loaded into the PCR system. Undigested DNA is shown as a control. (B) Cytosine methylation profiles assessed by bisulfite sequencing. The circles represent cytosine residues and are color coded according to the sequence context (red for CG, blue for CHG, and green for CHH). Solid circles indicate methylated cytosines. Each line represents the sequence of an individual clone. (C) Percentages of methylated cytosines in the TYLCV intergenic regions (IR). Student's t test was performed using the methylation values from individual clones. Double asterisks indicate a significant difference ( P
Figure Legend Snippet: NbHDA6 positively regulated the cytosine methylation of TYLCV. (A) Analysis of DNA methylation of the TYLCV genome by methylation-sensitive PCR. Genomic DNA was digested with HpaII, MspI, AluI, or McrBC and then loaded into the PCR system. Undigested DNA is shown as a control. (B) Cytosine methylation profiles assessed by bisulfite sequencing. The circles represent cytosine residues and are color coded according to the sequence context (red for CG, blue for CHG, and green for CHH). Solid circles indicate methylated cytosines. Each line represents the sequence of an individual clone. (C) Percentages of methylated cytosines in the TYLCV intergenic regions (IR). Student's t test was performed using the methylation values from individual clones. Double asterisks indicate a significant difference ( P

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

N. benthamiana HDA6 silenced lines show enhanced susceptibility to TYLCV infection. (A) Comparison of phenotypes of N. benthamiana plants in which the NbHDA6 gene was silenced (TRV-NbHDA6) and those of plants treated with a control silencing vector (TRV-GUS). (B) Relative NbHDA6 gene expression levels in VIGS and control N. benthamiana determined by real-time RT-PCR. TRV-NbHDA6-1, TRV-NbHDA6-2, and TRV-NbHDA6-3 represented three independent VIGS plants, and TRV-GUS represents the control plants. GAPDH was used as an internal control. Student's t test was performed, and double asterisks indicate a significant difference ( P
Figure Legend Snippet: N. benthamiana HDA6 silenced lines show enhanced susceptibility to TYLCV infection. (A) Comparison of phenotypes of N. benthamiana plants in which the NbHDA6 gene was silenced (TRV-NbHDA6) and those of plants treated with a control silencing vector (TRV-GUS). (B) Relative NbHDA6 gene expression levels in VIGS and control N. benthamiana determined by real-time RT-PCR. TRV-NbHDA6-1, TRV-NbHDA6-2, and TRV-NbHDA6-3 represented three independent VIGS plants, and TRV-GUS represents the control plants. GAPDH was used as an internal control. Student's t test was performed, and double asterisks indicate a significant difference ( P

Techniques Used: Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR

24) Product Images from "The Protein Neddylation Pathway in Trypanosoma brucei"

Article Title: The Protein Neddylation Pathway in Trypanosoma brucei

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.766741

Subcellular localization and knockdown of TbNedd8 in the procyclic form. A , subcellular localization of TbNedd8 in procyclic trypanosomes. TbNedd8 was endogenously tagged at the N terminus with EYFP, and the localization of EYFP-TbNedd8 was examined in paraformaldehyde-fixed intact cells and in detergent-extracted cytoskeletons. DIC , differential interference contrast. Scale bars = 5 μm. B , quantitative RT-PCR to monitor the mRNA level of TbNedd8 before and after RNAi induction for 24 h. C , Western blotting to detect the level of TbNedd8 and its conjugated proteins. TbNedd8 was tagged at the N terminus with a Myc epitope at one of the two endogenous loci in cells harboring the pSL-TbNedd8 RNAi construct. The crude cell lysate was immunoblotted with anti-Myc mAb to detect Myc-TbNedd8 and Myc-TbNedd8-conjugated proteins. The same blot was reprobed with anti-TbPSA6 pAb as the loading control. D , effect of TbNedd8 RNAi on cell proliferation. E , effect of TbNedd8 RNAi on global protein ubiquitination ( Ub ). Control and TbNedd8 RNAi cells were treated with MG-132 for 8 h and then analyzed by Western blotting with anti-ubiquitin mAb. The level of TbPSA6, T. brucei proteasome subunit α-6, served as the loading control. The histogram below the Western blots shows the quantitative data of the level of poly-ubiquitinated proteins, which was normalized against the level of TbPSA6. Error bars indicate standard deviation calculated from three independent experiments.
Figure Legend Snippet: Subcellular localization and knockdown of TbNedd8 in the procyclic form. A , subcellular localization of TbNedd8 in procyclic trypanosomes. TbNedd8 was endogenously tagged at the N terminus with EYFP, and the localization of EYFP-TbNedd8 was examined in paraformaldehyde-fixed intact cells and in detergent-extracted cytoskeletons. DIC , differential interference contrast. Scale bars = 5 μm. B , quantitative RT-PCR to monitor the mRNA level of TbNedd8 before and after RNAi induction for 24 h. C , Western blotting to detect the level of TbNedd8 and its conjugated proteins. TbNedd8 was tagged at the N terminus with a Myc epitope at one of the two endogenous loci in cells harboring the pSL-TbNedd8 RNAi construct. The crude cell lysate was immunoblotted with anti-Myc mAb to detect Myc-TbNedd8 and Myc-TbNedd8-conjugated proteins. The same blot was reprobed with anti-TbPSA6 pAb as the loading control. D , effect of TbNedd8 RNAi on cell proliferation. E , effect of TbNedd8 RNAi on global protein ubiquitination ( Ub ). Control and TbNedd8 RNAi cells were treated with MG-132 for 8 h and then analyzed by Western blotting with anti-ubiquitin mAb. The level of TbPSA6, T. brucei proteasome subunit α-6, served as the loading control. The histogram below the Western blots shows the quantitative data of the level of poly-ubiquitinated proteins, which was normalized against the level of TbPSA6. Error bars indicate standard deviation calculated from three independent experiments.

Techniques Used: Quantitative RT-PCR, Western Blot, Construct, Standard Deviation

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Clone Assay:

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Amplification:

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Article Snippet: .. The fragment containing CRISPR-Cas9 was ligated with a PCR fragment of pKLC21 that was amplified using a primer pair InF54 pKLC21 PCR-f and InF54 pKLC21 PCR-r to generate pKLC54. .. A spacer sequence against bla IMP-1 was inserted into the BsaI site of pKLC54 vector to obtain pKLC54_bla IMP-1_560 using Ligation high ver.

In Situ:

Article Title: Centrin3 in trypanosomes maintains the stability of a flagellar inner-arm dynein for cell motility
Article Snippet: .. Correct in situ tagging of one of the two TbCentrin3 alleles was confirmed by PCR and subsequent sequencing of the PCR fragment as well as Western blot with anti-GFP antibody (JL-8 clone, Clontech, 1:1000 dilution) to detect TbCentrin3::EYFP fusion protein or anti-Protein A polyclonal antibody (Sigma-Aldrich, 1:5000 dilution) to detect TbCentrin3::PTP fusion protein. .. DNA fragment corresponding to the C-terminal coding region of TbIAD5-1 was amplified by PCR (forward primer: 5’-GGCGAATTGGGTACCGGCTTGAGAGCATGTTCG-3’ and reverse primer: 5’-AGGATATTCCTTAAGATCATCTAGCATGCAGAG-3’) and cloned into the pC-3HA-PAC vector and transfected into the cell lines harboring the pZJM-TbIAD5-1 RNAi construct, pSL-TbCentrin3 RNAi construct, or the pC-TbCentrin3-EYFP-NEO construct.

Electroporation:

Article Title: Phosphatidylserine Synthase Controls Cell Elongation Especially in the Uppermost Internode in Rice by Regulation of Exocytosis
Article Snippet: .. The resulting PCR fragment was ligated into the Hind III and Pst I sites of pCAMBIA1305 (Cambia) using an infusion cloning kit (Clontech) to generate pCAMBIA1305-OsPSS-1, which was introduced into Agrobacterium tumefaciens strain EHA105 via electroporation and into the rice mutant as described previously [ ]. .. For RNAi, 539-bp fragments of OsPSS-1 were amplified using primer pairs OsPSS-1-RNAi1-KpnI-F/R and OsPSS-1-RNAi2-BamHI-F/R ( ).

Mutagenesis:

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Construct:

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Purification:

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Article Snippet: .. Approximately 1–5 μg of the purified PCR fragment was transformed into the yIG397 yeast strain using the lithium acetate procedure according to the Yeast Transformation System Kit by CLONTECH (Palo Alto, CA). .. After transformation the cells were plated on either YPD agar plates containing 200μg/mL G418 or –HIS plates.

Sequencing:

Article Title: The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant
Article Snippet: .. The drnf1 sequence was amplified by PCR with the primers drnf1 -F and drnf1 -R. The PCR fragment was ligated into pMD18-T vector (Takara, Dalian, China) for sequencing. .. The resulting plasmid was inserted with an omega -PrbcL fragment containing spectinomycin-resistant gene and rbcL promoter in the Sal I site (ahead of the drnf1 gene).

Article Title: Centrin3 in trypanosomes maintains the stability of a flagellar inner-arm dynein for cell motility
Article Snippet: .. Correct in situ tagging of one of the two TbCentrin3 alleles was confirmed by PCR and subsequent sequencing of the PCR fragment as well as Western blot with anti-GFP antibody (JL-8 clone, Clontech, 1:1000 dilution) to detect TbCentrin3::EYFP fusion protein or anti-Protein A polyclonal antibody (Sigma-Aldrich, 1:5000 dilution) to detect TbCentrin3::PTP fusion protein. .. DNA fragment corresponding to the C-terminal coding region of TbIAD5-1 was amplified by PCR (forward primer: 5’-GGCGAATTGGGTACCGGCTTGAGAGCATGTTCG-3’ and reverse primer: 5’-AGGATATTCCTTAAGATCATCTAGCATGCAGAG-3’) and cloned into the pC-3HA-PAC vector and transfected into the cell lines harboring the pZJM-TbIAD5-1 RNAi construct, pSL-TbCentrin3 RNAi construct, or the pC-TbCentrin3-EYFP-NEO construct.

CRISPR:

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria
Article Snippet: .. The fragment containing CRISPR-Cas9 was ligated with a PCR fragment of pKLC21 that was amplified using a primer pair InF54 pKLC21 PCR-f and InF54 pKLC21 PCR-r to generate pKLC54. .. A spacer sequence against bla IMP-1 was inserted into the BsaI site of pKLC54 vector to obtain pKLC54_bla IMP-1_560 using Ligation high ver.

Polymerase Chain Reaction:

Article Title: The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant
Article Snippet: .. The drnf1 sequence was amplified by PCR with the primers drnf1 -F and drnf1 -R. The PCR fragment was ligated into pMD18-T vector (Takara, Dalian, China) for sequencing. .. The resulting plasmid was inserted with an omega -PrbcL fragment containing spectinomycin-resistant gene and rbcL promoter in the Sal I site (ahead of the drnf1 gene).

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria
Article Snippet: .. The fragment containing CRISPR-Cas9 was ligated with a PCR fragment of pKLC21 that was amplified using a primer pair InF54 pKLC21 PCR-f and InF54 pKLC21 PCR-r to generate pKLC54. .. A spacer sequence against bla IMP-1 was inserted into the BsaI site of pKLC54 vector to obtain pKLC54_bla IMP-1_560 using Ligation high ver.

Article Title: The role of base excision repair genes OGG1, APN1 and APN2 in benzo[a]pyrene-7,8-dione induced p53 mutagenesis
Article Snippet: .. Approximately 1–5 μg of the purified PCR fragment was transformed into the yIG397 yeast strain using the lithium acetate procedure according to the Yeast Transformation System Kit by CLONTECH (Palo Alto, CA). .. After transformation the cells were plated on either YPD agar plates containing 200μg/mL G418 or –HIS plates.

Article Title: Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9
Article Snippet: .. The PCR fragment was ligated with linker-EGFP excised from pCAG-PSD-95-EGFP with Bam HI and Not I, using the In-Fusion HD Cloning Kit (Takara Bio, Shiga, Japan) to generate PSD-95 targeting vector (EGFP). .. PSD-95 targeting vector (mCherry) was constructed by digesting PSD-95 targeting vector (EGFP) with Bam HI and Not I, and replacing the coding region of EGFP with that of mCherry excised from pCAG-Syp-mCherry with Bam HI/Not I.

Article Title: Engineering of Fatty Acid Synthases (FASs) to Boost the Production of Medium-Chain Fatty Acids (MCFAs) in Mucor circinelloides
Article Snippet: .. The aforesaid primers exhibited homologous sequences (i.e., 28 bp) to the Xho I restriction sites in plasmid pMAT1552-pyrF, and the PCR fragment was finally cloned into plasmid pMAT1552-pyrF using the restriction endonuclease Xho I to construct the recombinant plasmids i.e., pMAT1552-pyrF-TE-01, pMAT1552-pyrF-TE-02, pMAT1552-pyrF-TE-03, pMAT1552-pyrF-TE-04 (One-Step Cloning Kit from Takara Bio USA, Inc., CA, USA). ..

Article Title: Phosphatidylserine Synthase Controls Cell Elongation Especially in the Uppermost Internode in Rice by Regulation of Exocytosis
Article Snippet: .. The resulting PCR fragment was ligated into the Hind III and Pst I sites of pCAMBIA1305 (Cambia) using an infusion cloning kit (Clontech) to generate pCAMBIA1305-OsPSS-1, which was introduced into Agrobacterium tumefaciens strain EHA105 via electroporation and into the rice mutant as described previously [ ]. .. For RNAi, 539-bp fragments of OsPSS-1 were amplified using primer pairs OsPSS-1-RNAi1-KpnI-F/R and OsPSS-1-RNAi2-BamHI-F/R ( ).

Article Title: A 40 kDa protein of the inner membrane is the mitochondrial calcium uniporter
Article Snippet: .. - For the cloning of MICU1 in pEGFP-N1: fw: 5′-CCTCGAGATGTTTCGTCTTAACACCCT-3′ rv: 5′-CGGATCCCGTTTGGGCAGAGCAAAGTCCC-3′ The PCR fragment was cloned into XhoI and BamHI sites in pEGFP-N1 (Clontech). ..

Article Title: Centrin3 in trypanosomes maintains the stability of a flagellar inner-arm dynein for cell motility
Article Snippet: .. Correct in situ tagging of one of the two TbCentrin3 alleles was confirmed by PCR and subsequent sequencing of the PCR fragment as well as Western blot with anti-GFP antibody (JL-8 clone, Clontech, 1:1000 dilution) to detect TbCentrin3::EYFP fusion protein or anti-Protein A polyclonal antibody (Sigma-Aldrich, 1:5000 dilution) to detect TbCentrin3::PTP fusion protein. .. DNA fragment corresponding to the C-terminal coding region of TbIAD5-1 was amplified by PCR (forward primer: 5’-GGCGAATTGGGTACCGGCTTGAGAGCATGTTCG-3’ and reverse primer: 5’-AGGATATTCCTTAAGATCATCTAGCATGCAGAG-3’) and cloned into the pC-3HA-PAC vector and transfected into the cell lines harboring the pZJM-TbIAD5-1 RNAi construct, pSL-TbCentrin3 RNAi construct, or the pC-TbCentrin3-EYFP-NEO construct.

Western Blot:

Article Title: Centrin3 in trypanosomes maintains the stability of a flagellar inner-arm dynein for cell motility
Article Snippet: .. Correct in situ tagging of one of the two TbCentrin3 alleles was confirmed by PCR and subsequent sequencing of the PCR fragment as well as Western blot with anti-GFP antibody (JL-8 clone, Clontech, 1:1000 dilution) to detect TbCentrin3::EYFP fusion protein or anti-Protein A polyclonal antibody (Sigma-Aldrich, 1:5000 dilution) to detect TbCentrin3::PTP fusion protein. .. DNA fragment corresponding to the C-terminal coding region of TbIAD5-1 was amplified by PCR (forward primer: 5’-GGCGAATTGGGTACCGGCTTGAGAGCATGTTCG-3’ and reverse primer: 5’-AGGATATTCCTTAAGATCATCTAGCATGCAGAG-3’) and cloned into the pC-3HA-PAC vector and transfected into the cell lines harboring the pZJM-TbIAD5-1 RNAi construct, pSL-TbCentrin3 RNAi construct, or the pC-TbCentrin3-EYFP-NEO construct.

Transformation Assay:

Article Title: The role of base excision repair genes OGG1, APN1 and APN2 in benzo[a]pyrene-7,8-dione induced p53 mutagenesis
Article Snippet: .. Approximately 1–5 μg of the purified PCR fragment was transformed into the yIG397 yeast strain using the lithium acetate procedure according to the Yeast Transformation System Kit by CLONTECH (Palo Alto, CA). .. After transformation the cells were plated on either YPD agar plates containing 200μg/mL G418 or –HIS plates.

Recombinant:

Article Title: Engineering of Fatty Acid Synthases (FASs) to Boost the Production of Medium-Chain Fatty Acids (MCFAs) in Mucor circinelloides
Article Snippet: .. The aforesaid primers exhibited homologous sequences (i.e., 28 bp) to the Xho I restriction sites in plasmid pMAT1552-pyrF, and the PCR fragment was finally cloned into plasmid pMAT1552-pyrF using the restriction endonuclease Xho I to construct the recombinant plasmids i.e., pMAT1552-pyrF-TE-01, pMAT1552-pyrF-TE-02, pMAT1552-pyrF-TE-03, pMAT1552-pyrF-TE-04 (One-Step Cloning Kit from Takara Bio USA, Inc., CA, USA). ..

Plasmid Preparation:

Article Title: The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant
Article Snippet: .. The drnf1 sequence was amplified by PCR with the primers drnf1 -F and drnf1 -R. The PCR fragment was ligated into pMD18-T vector (Takara, Dalian, China) for sequencing. .. The resulting plasmid was inserted with an omega -PrbcL fragment containing spectinomycin-resistant gene and rbcL promoter in the Sal I site (ahead of the drnf1 gene).

Article Title: Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9
Article Snippet: .. The PCR fragment was ligated with linker-EGFP excised from pCAG-PSD-95-EGFP with Bam HI and Not I, using the In-Fusion HD Cloning Kit (Takara Bio, Shiga, Japan) to generate PSD-95 targeting vector (EGFP). .. PSD-95 targeting vector (mCherry) was constructed by digesting PSD-95 targeting vector (EGFP) with Bam HI and Not I, and replacing the coding region of EGFP with that of mCherry excised from pCAG-Syp-mCherry with Bam HI/Not I.

Article Title: Engineering of Fatty Acid Synthases (FASs) to Boost the Production of Medium-Chain Fatty Acids (MCFAs) in Mucor circinelloides
Article Snippet: .. The aforesaid primers exhibited homologous sequences (i.e., 28 bp) to the Xho I restriction sites in plasmid pMAT1552-pyrF, and the PCR fragment was finally cloned into plasmid pMAT1552-pyrF using the restriction endonuclease Xho I to construct the recombinant plasmids i.e., pMAT1552-pyrF-TE-01, pMAT1552-pyrF-TE-02, pMAT1552-pyrF-TE-03, pMAT1552-pyrF-TE-04 (One-Step Cloning Kit from Takara Bio USA, Inc., CA, USA). ..

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  • 92
    TaKaRa pcr amplified fragments
    Colocalization of endogenous proteins with the H/ACA RNA transcription site. (A) Double fluorescence of a fixed and permeabilized <t>E3</t> cell after induction of the transgene and immunostained for endogenous NAP57 (panel 1) and for the H/ACA RNA transcription site by FISH with the probe to intron 1 (panel 2). Panel 3 shows a merge of the two in pseudocolor. Panel 4 depicts the corresponding phase-contrast image outlining, with a box indicating the area containing the transcription site, which is enlarged in the insets. (B–D) Same as in A, except immunostained for the endogenous proteins indicated on the left, and the transcription site was identified by the fluorescence of transfected MS2-RFP (panel 2). (E) Bar diagram of the percentage of cells, with the indicated proteins observed at the site of H/ACA RNA transcription. The following numbers of cells with a transcription site (in parentheses) were counted for each protein: NAP57 (150); NOP10 (101); NHP2 (101); GAR1 (104); and NAF1 (159). Questionable colocalizations were discounted. (F) ChIPs of the GFP-fusion constructs indicated on top after their transient transfection into E3 cells and induction of the transgene. Immunoprecipitations were performed with GFP antibodies. Ethidium bromide stain of DNA amplified by <t>PCR,</t> with primers to the regions of the transgene indicated on the right and separated on agarose gels. Amplifications of the input (I; odd lanes) and the ChIPs (even lanes) are shown. (G) Same as in B and C, except stained for the Cajal body marker protein coilin.
    Pcr Amplified Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa pcr fragments
    <t>Sun3</t> is a testis specific protein showing a postmeiotic expression profile. (A) Total RNA was isolated from different tissues and used for <t>RT-PCR.</t> cDNA was amplified using Sun3-specific primers and a GAPDH-PCR served as control for RNA fidelity. (B) Samples from the same tissues were homogenized in SDS sample buffer and separated by SDS-PAGE (5×10 5 cells; 30 µg protein/lane). Sun3 protein was detected with an affinity-purified anti-Sun3 antiserum (Arrow). (C) Total RNA was isolated from testicular cells from pubertal mice of increasing ages (8-25 days p.p.) and used for RT-PCR. cDNA was amplified using primers for Sun3, Cage1 (postmeiotic expression) and Sycp3 (meiotic expression). (D) To analyze stage-specific expression of protein Sun3 testes from pubertal mice were homogenized in SDS sample buffer and separated by SDS-PAGE (4×10 5 cells/lane). Sun3 protein was detected with affinity-purified anti-Sun3 antiserum. For comparison pAbs against Lamin B3 and Cage1 were used. (E) Localization of Sun3 within seminiferous tubules was analyzed by indirect immunofluorescence microscopy. Testis paraffin sections of adult mice were stained using affinity-purified anti-Sun3 antiserum (green) and mAb 13d4 against LAP2 (red). DNA was labeled with 33258-Hoechst (blue). Sun3 was only detectable in spermatids (Sp) but completely absent from somatic cells (S), spermatogonia (Sg) and spermatocytes (Sc). Scale bar: 15 µm.
    Pcr Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 600 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa eco ri pcr amplified fragment
    Interaction between calreticulin and <t>TMV</t> MP and effect of calreticulin overexpression on TMV MP subcellular localization. A, Interaction in yeast two-hybrid system. Lane 1, AtCRT1 + TMV MP; lane 2, AtCRT1 + lamin C; lane 3, AtCRT1 lacking signal peptide + TMV MP. B, <t>RT-PCR</t> analysis of ZmCRT1 overexpression in transgenic N. benthamiana . Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Top and bottom bands correspond to the ZmCRT1- and actin-specific products. Fragment size in base pairs is indicated on the right. C, Western-blot analysis of ZmCRT1 overexpression in transgenic plants. Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Molecular mass expressed in thousands of Daltons is indicated on the right. D, Interaction in renatured-blot overlay assay. Lane 1, Plants transgenic for ZmCRT1 + TMV MP; lane 2, wild-type plants + TMV MP; lane 3, plants transgenic for ZmCRT1 + VirD2. Arrowhead indicates the position of calreticulin. Molecular mass expressed in thousands of Daltons is indicated on the right. E, GFP-TMV MP transiently expressed in wild-type N. benthamiana . F, GFP-TMV MP transiently expressed in transgenic N. benthamiana plants overexpressing ZmCRT1. GFP signal is in green; plastid autofluorescence is in red. Images in E and F are projections of several confocal sections; plastid autofluorescence in E was filtered out. Bars = 10 μ m.
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    Colocalization of endogenous proteins with the H/ACA RNA transcription site. (A) Double fluorescence of a fixed and permeabilized E3 cell after induction of the transgene and immunostained for endogenous NAP57 (panel 1) and for the H/ACA RNA transcription site by FISH with the probe to intron 1 (panel 2). Panel 3 shows a merge of the two in pseudocolor. Panel 4 depicts the corresponding phase-contrast image outlining, with a box indicating the area containing the transcription site, which is enlarged in the insets. (B–D) Same as in A, except immunostained for the endogenous proteins indicated on the left, and the transcription site was identified by the fluorescence of transfected MS2-RFP (panel 2). (E) Bar diagram of the percentage of cells, with the indicated proteins observed at the site of H/ACA RNA transcription. The following numbers of cells with a transcription site (in parentheses) were counted for each protein: NAP57 (150); NOP10 (101); NHP2 (101); GAR1 (104); and NAF1 (159). Questionable colocalizations were discounted. (F) ChIPs of the GFP-fusion constructs indicated on top after their transient transfection into E3 cells and induction of the transgene. Immunoprecipitations were performed with GFP antibodies. Ethidium bromide stain of DNA amplified by PCR, with primers to the regions of the transgene indicated on the right and separated on agarose gels. Amplifications of the input (I; odd lanes) and the ChIPs (even lanes) are shown. (G) Same as in B and C, except stained for the Cajal body marker protein coilin.

    Journal: The Journal of Cell Biology

    Article Title: Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells

    doi: 10.1083/jcb.200601105

    Figure Lengend Snippet: Colocalization of endogenous proteins with the H/ACA RNA transcription site. (A) Double fluorescence of a fixed and permeabilized E3 cell after induction of the transgene and immunostained for endogenous NAP57 (panel 1) and for the H/ACA RNA transcription site by FISH with the probe to intron 1 (panel 2). Panel 3 shows a merge of the two in pseudocolor. Panel 4 depicts the corresponding phase-contrast image outlining, with a box indicating the area containing the transcription site, which is enlarged in the insets. (B–D) Same as in A, except immunostained for the endogenous proteins indicated on the left, and the transcription site was identified by the fluorescence of transfected MS2-RFP (panel 2). (E) Bar diagram of the percentage of cells, with the indicated proteins observed at the site of H/ACA RNA transcription. The following numbers of cells with a transcription site (in parentheses) were counted for each protein: NAP57 (150); NOP10 (101); NHP2 (101); GAR1 (104); and NAF1 (159). Questionable colocalizations were discounted. (F) ChIPs of the GFP-fusion constructs indicated on top after their transient transfection into E3 cells and induction of the transgene. Immunoprecipitations were performed with GFP antibodies. Ethidium bromide stain of DNA amplified by PCR, with primers to the regions of the transgene indicated on the right and separated on agarose gels. Amplifications of the input (I; odd lanes) and the ChIPs (even lanes) are shown. (G) Same as in B and C, except stained for the Cajal body marker protein coilin.

    Article Snippet: The pTet-globin-snoE3-CFP-18MS2-2 (for E3 cells) and pTet-globin-CFP-18MS2-2 (for E3-minus cells) were generated by sequentially inserting the following, mostly PCR-amplified fragments, into pCMV-globin ( ): CFP with a COOH-terminal SKL tripeptide from pECFP-N1 (CLONTECH Laboratories, Inc.); 18 MS2 repeats from pSL-24X ( ; six repeats were lost because of recombination in Escherichia coli ); Tet promoter from pTRE-2 (CLONTECH Laboratories, Inc.) replacing the CMV promoter; and, after excision of the neomycin-resistance gene, rat snoRNA E3 from pTM105 ( ).

    Techniques: Fluorescence, Fluorescence In Situ Hybridization, Transfection, Construct, Staining, Amplification, Polymerase Chain Reaction, Marker

    Sun3 is a testis specific protein showing a postmeiotic expression profile. (A) Total RNA was isolated from different tissues and used for RT-PCR. cDNA was amplified using Sun3-specific primers and a GAPDH-PCR served as control for RNA fidelity. (B) Samples from the same tissues were homogenized in SDS sample buffer and separated by SDS-PAGE (5×10 5 cells; 30 µg protein/lane). Sun3 protein was detected with an affinity-purified anti-Sun3 antiserum (Arrow). (C) Total RNA was isolated from testicular cells from pubertal mice of increasing ages (8-25 days p.p.) and used for RT-PCR. cDNA was amplified using primers for Sun3, Cage1 (postmeiotic expression) and Sycp3 (meiotic expression). (D) To analyze stage-specific expression of protein Sun3 testes from pubertal mice were homogenized in SDS sample buffer and separated by SDS-PAGE (4×10 5 cells/lane). Sun3 protein was detected with affinity-purified anti-Sun3 antiserum. For comparison pAbs against Lamin B3 and Cage1 were used. (E) Localization of Sun3 within seminiferous tubules was analyzed by indirect immunofluorescence microscopy. Testis paraffin sections of adult mice were stained using affinity-purified anti-Sun3 antiserum (green) and mAb 13d4 against LAP2 (red). DNA was labeled with 33258-Hoechst (blue). Sun3 was only detectable in spermatids (Sp) but completely absent from somatic cells (S), spermatogonia (Sg) and spermatocytes (Sc). Scale bar: 15 µm.

    Journal: PLoS ONE

    Article Title: Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes

    doi: 10.1371/journal.pone.0012072

    Figure Lengend Snippet: Sun3 is a testis specific protein showing a postmeiotic expression profile. (A) Total RNA was isolated from different tissues and used for RT-PCR. cDNA was amplified using Sun3-specific primers and a GAPDH-PCR served as control for RNA fidelity. (B) Samples from the same tissues were homogenized in SDS sample buffer and separated by SDS-PAGE (5×10 5 cells; 30 µg protein/lane). Sun3 protein was detected with an affinity-purified anti-Sun3 antiserum (Arrow). (C) Total RNA was isolated from testicular cells from pubertal mice of increasing ages (8-25 days p.p.) and used for RT-PCR. cDNA was amplified using primers for Sun3, Cage1 (postmeiotic expression) and Sycp3 (meiotic expression). (D) To analyze stage-specific expression of protein Sun3 testes from pubertal mice were homogenized in SDS sample buffer and separated by SDS-PAGE (4×10 5 cells/lane). Sun3 protein was detected with affinity-purified anti-Sun3 antiserum. For comparison pAbs against Lamin B3 and Cage1 were used. (E) Localization of Sun3 within seminiferous tubules was analyzed by indirect immunofluorescence microscopy. Testis paraffin sections of adult mice were stained using affinity-purified anti-Sun3 antiserum (green) and mAb 13d4 against LAP2 (red). DNA was labeled with 33258-Hoechst (blue). Sun3 was only detectable in spermatids (Sp) but completely absent from somatic cells (S), spermatogonia (Sg) and spermatocytes (Sc). Scale bar: 15 µm.

    Article Snippet: To generate Myc-tagged constructs for immunoprecipitation, PCR fragments of Sun3 coding for the full-length protein (Sun3FL: Sun3-5’Eco; Sun3-3’Sal) and Sun1 coding for amino acids 501–913 (Sun1TM+Lum: Sun1IP-5’Eco; Sun1IP-3’Sal) were cloned into pCMV-Myc vector (Clontech Laboratories).

    Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, SDS Page, Affinity Purification, Mouse Assay, Immunofluorescence, Microscopy, Staining, Labeling

    Raver1 is widely expressed as revealed by Northern blot analysis . Two cDNA fragments (bp 1–1326 and 1327–2257) comprising the whole ORF were amplified by PCR using DIG-labeled nucleotides and hybridized with total RNA on a mouse multiple tissue Northern (A) and a mouse RNA master (B) blot. Note that raver1 mRNA is detected in all adult mouse tissues tested, although in varying intensities (A, 1, heart; 2, brain; 3, lung; 4, spleen; 5, liver; 6, kidney; 7, skeletal muscle; 8, testis) and embryonic stages 7–17 (B, indicated above samples). Equal RNA load is demonstrated by probing for β-actin mRNA.

    Journal: The Journal of Cell Biology

    Article Title: Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins

    doi: 10.1083/jcb.200105044

    Figure Lengend Snippet: Raver1 is widely expressed as revealed by Northern blot analysis . Two cDNA fragments (bp 1–1326 and 1327–2257) comprising the whole ORF were amplified by PCR using DIG-labeled nucleotides and hybridized with total RNA on a mouse multiple tissue Northern (A) and a mouse RNA master (B) blot. Note that raver1 mRNA is detected in all adult mouse tissues tested, although in varying intensities (A, 1, heart; 2, brain; 3, lung; 4, spleen; 5, liver; 6, kidney; 7, skeletal muscle; 8, testis) and embryonic stages 7–17 (B, indicated above samples). Equal RNA load is demonstrated by probing for β-actin mRNA.

    Article Snippet: Northern blotting Two PCR fragments comprising the entire ORF of raver1 were DIG labeled as described above and used to probe a mouse multiple tissue Northern blot and a mouse master blot, both purchased from CLONTECH.

    Techniques: Northern Blot, Amplification, Polymerase Chain Reaction, Labeling

    Interaction between calreticulin and TMV MP and effect of calreticulin overexpression on TMV MP subcellular localization. A, Interaction in yeast two-hybrid system. Lane 1, AtCRT1 + TMV MP; lane 2, AtCRT1 + lamin C; lane 3, AtCRT1 lacking signal peptide + TMV MP. B, RT-PCR analysis of ZmCRT1 overexpression in transgenic N. benthamiana . Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Top and bottom bands correspond to the ZmCRT1- and actin-specific products. Fragment size in base pairs is indicated on the right. C, Western-blot analysis of ZmCRT1 overexpression in transgenic plants. Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Molecular mass expressed in thousands of Daltons is indicated on the right. D, Interaction in renatured-blot overlay assay. Lane 1, Plants transgenic for ZmCRT1 + TMV MP; lane 2, wild-type plants + TMV MP; lane 3, plants transgenic for ZmCRT1 + VirD2. Arrowhead indicates the position of calreticulin. Molecular mass expressed in thousands of Daltons is indicated on the right. E, GFP-TMV MP transiently expressed in wild-type N. benthamiana . F, GFP-TMV MP transiently expressed in transgenic N. benthamiana plants overexpressing ZmCRT1. GFP signal is in green; plastid autofluorescence is in red. Images in E and F are projections of several confocal sections; plastid autofluorescence in E was filtered out. Bars = 10 μ m.

    Journal: Plant Physiology

    Article Title: Effects of Calreticulin on Viral Cell-to-Cell Movement 1

    doi: 10.1104/pp.105.064386

    Figure Lengend Snippet: Interaction between calreticulin and TMV MP and effect of calreticulin overexpression on TMV MP subcellular localization. A, Interaction in yeast two-hybrid system. Lane 1, AtCRT1 + TMV MP; lane 2, AtCRT1 + lamin C; lane 3, AtCRT1 lacking signal peptide + TMV MP. B, RT-PCR analysis of ZmCRT1 overexpression in transgenic N. benthamiana . Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Top and bottom bands correspond to the ZmCRT1- and actin-specific products. Fragment size in base pairs is indicated on the right. C, Western-blot analysis of ZmCRT1 overexpression in transgenic plants. Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Molecular mass expressed in thousands of Daltons is indicated on the right. D, Interaction in renatured-blot overlay assay. Lane 1, Plants transgenic for ZmCRT1 + TMV MP; lane 2, wild-type plants + TMV MP; lane 3, plants transgenic for ZmCRT1 + VirD2. Arrowhead indicates the position of calreticulin. Molecular mass expressed in thousands of Daltons is indicated on the right. E, GFP-TMV MP transiently expressed in wild-type N. benthamiana . F, GFP-TMV MP transiently expressed in transgenic N. benthamiana plants overexpressing ZmCRT1. GFP signal is in green; plastid autofluorescence is in red. Images in E and F are projections of several confocal sections; plastid autofluorescence in E was filtered out. Bars = 10 μ m.

    Article Snippet: For a translational fusion of TMV MP to the N terminus of CFP (TMV MP-CFP), the coding sequence of TMV MP was first cloned as a Sal I- Eco RI PCR-amplified fragment into the Xho I- Eco RI sites of pSAT1-MCS , and, into the Sal I- Bam HI sites of the resulting construct, the coding sequence of CFP (derived from pECFP-N1; CLONTECH Laboratories, Mountain View, CA) was then cloned as a Sal I-BglII PCR-amplified fragment.

    Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Western Blot, Overlay Assay