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  • 99
    Millipore single stranded dna templates
    Single Stranded Dna Templates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    single stranded dna templates - by Bioz Stars, 2020-04
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    90
    Thermo Fisher dna template
    Standard curve demonstrating the range of threshold cycle (Ct) values plotted versus genome equivalents (GE) of Brucella pinnipedialis strain B04-0821 per <t>PCR</t> reaction volume (1.5 µL of <t>DNA).</t> The regression line represents data that were in the linear range. Each point represents the mean value for triplicate runs at each dilution.
    Dna Template, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 7589 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega dna template
    Standard curve demonstrating the range of threshold cycle (Ct) values plotted versus genome equivalents (GE) of Brucella pinnipedialis strain B04-0821 per <t>PCR</t> reaction volume (1.5 µL of <t>DNA).</t> The regression line represents data that were in the linear range. Each point represents the mean value for triplicate runs at each dilution.
    Dna Template, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3468 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega dna templates
    Effect of the mutated AP-1 sites on the regulation of the TF promoter by <t>PML/RARα.</t> EMSA analysis for the interaction of PML/RARα with the TF promoter was performed using radiolabeled <t>DNA</t> probes spanning from position −250 to −196
    Dna Templates, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 832 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dna templates
    RT progression assay determining G-quartet formation in the Sp1 binding region. Cation-dependent pausing of reverse transcription at the guanine-rich elements in the U3 region was analyzed with <t>RNA</t> and <t>DNA</t> templates. A fragment of the RNA/DNA sequence of the Sp1 binding region is shown (top) with G-rich elements (shaded). Strong pauses of the RT near G-rich elements were observed in the presence of 50 mM of KCl, but not LiCl, indicating that these elements are involved in the formation of structure, which is stabilized by potassium ions but destabilized by lithium ions. This is indicative of a G-quadruplex. The cation-independent RT pauses are likely caused by hairpin structures. DNA primer, P; DNA marker, M; KCl, K + ; LiCl, Li + .
    Dna Templates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3027 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATUM template dna
    <t>PCR</t> amplification of the nuc gene of S. aureus . Lane 1: 100 bp <t>DNA</t> ladder. Lane 5: Amplified PCR product
    Template Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 99/100, based on 1683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    template dna - by Bioz Stars, 2020-04
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    99
    Millipore template dna
    Dendrogram showing the relatedness of E. coli strains collected from the 72 pigs isolated in four pens as determined by <t>REP-PCR.</t> A condensed dendrogram (using a 60% cut-off value) is shown and contains 20 major clusters. <t>DNA</t> fingerprint similarities were calculated by the curve-based Pearson coefficient, and dendrograms were generated by UPGMA. Dominant profiles previously detected in this farm [ 11 ] are indicated between brackets
    Template Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    DSMZ template dna
    Dendrogram showing the relatedness of E. coli strains collected from the 72 pigs isolated in four pens as determined by <t>REP-PCR.</t> A condensed dendrogram (using a 60% cut-off value) is shown and contains 20 major clusters. <t>DNA</t> fingerprint similarities were calculated by the curve-based Pearson coefficient, and dendrograms were generated by UPGMA. Dominant profiles previously detected in this farm [ 11 ] are indicated between brackets
    Template Dna, supplied by DSMZ, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GE Healthcare template dna
    Mouse <t>Mbnl3</t> knockout characterization and Mbnl3 muscle RNA targets in vivo . ( A ) Genomic <t>DNA</t> blot of wild-type ( Mbnl3 + ) and Mbnl3 isoform knockout ( Mbnl3 ΔE2 ), showing the reduction in Kpn I fragment length in Mbnl3 ΔE2 knockouts. ( B ) RT–PCR
    Template Dna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa template dna
    Inverse <t>PCR</t> (iPCR) and sequencing of the boundary region surrounding the Mk + 1.3 locus. ( a ) Agarose gel electrophoresis of nested PCR products stained with ethidium bromide. The gel is the full length gel and lanes have not been cropped or stitched. ( b ) Chromatograms generated from direct sequencing of the iPCR products. Black and red letters above the chromatograms indicate the sequences of chromosomal positions chr 7: 62,416,891–62,416,891 and chr 7: 62,268,615–62,268,638 (on the University of California, Santa Cruz, genome browser (GRCm38/mm10)), respectively. W: wild-type <t>DNA,</t> Tg: hemizygous transgenic DNA, M: molecular size marker.
    Template Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 3940 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Illumina Inc dna templates
    Procedure for the AAV Barcode-Seq analysis. We applied <t>DNA-barcoded</t> AAV library stocks to tissue culture cells for receptor binding and in vitro transduction assays. We injected them into mice to investigate blood clearance rate, tissue tropism, reactivity to neutralizing antibodies and anti-AAV neutralizing antibody epitope mapping. We then collected various samples, extracted DNA, PCR-amplified AAV clone-specific Virus Bar Codes (VBCs) using Sample-specific Bar Code (SBC)-attached PCR primers. We mixed all the VBC–PCR amplicons in a pool, and sequenced them on the <t>Illumina</t> platform. We then converted raw sequence read number data to PD values by a computational algorithm.
    Dna Templates, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 96/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Microsynth double stranded dna templates
    Procedure for the AAV Barcode-Seq analysis. We applied <t>DNA-barcoded</t> AAV library stocks to tissue culture cells for receptor binding and in vitro transduction assays. We injected them into mice to investigate blood clearance rate, tissue tropism, reactivity to neutralizing antibodies and anti-AAV neutralizing antibody epitope mapping. We then collected various samples, extracted DNA, PCR-amplified AAV clone-specific Virus Bar Codes (VBCs) using Sample-specific Bar Code (SBC)-attached PCR primers. We mixed all the VBC–PCR amplicons in a pool, and sequenced them on the <t>Illumina</t> platform. We then converted raw sequence read number data to PD values by a computational algorithm.
    Double Stranded Dna Templates, supplied by Microsynth, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    5 PRIME template dna
    Procedure for the AAV Barcode-Seq analysis. We applied <t>DNA-barcoded</t> AAV library stocks to tissue culture cells for receptor binding and in vitro transduction assays. We injected them into mice to investigate blood clearance rate, tissue tropism, reactivity to neutralizing antibodies and anti-AAV neutralizing antibody epitope mapping. We then collected various samples, extracted DNA, PCR-amplified AAV clone-specific Virus Bar Codes (VBCs) using Sample-specific Bar Code (SBC)-attached PCR primers. We mixed all the VBC–PCR amplicons in a pool, and sequenced them on the <t>Illumina</t> platform. We then converted raw sequence read number data to PD values by a computational algorithm.
    Template Dna, supplied by 5 PRIME, used in various techniques. Bioz Stars score: 92/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Pacific Biosciences template dna
    Procedure for the AAV Barcode-Seq analysis. We applied <t>DNA-barcoded</t> AAV library stocks to tissue culture cells for receptor binding and in vitro transduction assays. We injected them into mice to investigate blood clearance rate, tissue tropism, reactivity to neutralizing antibodies and anti-AAV neutralizing antibody epitope mapping. We then collected various samples, extracted DNA, PCR-amplified AAV clone-specific Virus Bar Codes (VBCs) using Sample-specific Bar Code (SBC)-attached PCR primers. We mixed all the VBC–PCR amplicons in a pool, and sequenced them on the <t>Illumina</t> platform. We then converted raw sequence read number data to PD values by a computational algorithm.
    Template Dna, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Glen Research dna templates
    Procedure for the AAV Barcode-Seq analysis. We applied <t>DNA-barcoded</t> AAV library stocks to tissue culture cells for receptor binding and in vitro transduction assays. We injected them into mice to investigate blood clearance rate, tissue tropism, reactivity to neutralizing antibodies and anti-AAV neutralizing antibody epitope mapping. We then collected various samples, extracted DNA, PCR-amplified AAV clone-specific Virus Bar Codes (VBCs) using Sample-specific Bar Code (SBC)-attached PCR primers. We mixed all the VBC–PCR amplicons in a pool, and sequenced them on the <t>Illumina</t> platform. We then converted raw sequence read number data to PD values by a computational algorithm.
    Dna Templates, supplied by Glen Research, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    OriGene dna templates
    Procedure for the AAV Barcode-Seq analysis. We applied <t>DNA-barcoded</t> AAV library stocks to tissue culture cells for receptor binding and in vitro transduction assays. We injected them into mice to investigate blood clearance rate, tissue tropism, reactivity to neutralizing antibodies and anti-AAV neutralizing antibody epitope mapping. We then collected various samples, extracted DNA, PCR-amplified AAV clone-specific Virus Bar Codes (VBCs) using Sample-specific Bar Code (SBC)-attached PCR primers. We mixed all the VBC–PCR amplicons in a pool, and sequenced them on the <t>Illumina</t> platform. We then converted raw sequence read number data to PD values by a computational algorithm.
    Dna Templates, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    OriGene template dna
    Procedure for the AAV Barcode-Seq analysis. We applied <t>DNA-barcoded</t> AAV library stocks to tissue culture cells for receptor binding and in vitro transduction assays. We injected them into mice to investigate blood clearance rate, tissue tropism, reactivity to neutralizing antibodies and anti-AAV neutralizing antibody epitope mapping. We then collected various samples, extracted DNA, PCR-amplified AAV clone-specific Virus Bar Codes (VBCs) using Sample-specific Bar Code (SBC)-attached PCR primers. We mixed all the VBC–PCR amplicons in a pool, and sequenced them on the <t>Illumina</t> platform. We then converted raw sequence read number data to PD values by a computational algorithm.
    Template Dna, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc template dna
    Reprogramming of MEFs with SeVdp vectors installed with reprogramming genes. A , the genome structure of SeVdp( c-Myc / <t>Klf4</t> / Oct4 / Sox2 ) is shown. B , shown is genome structure of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ), coexisting in a single cell, is shown. C and D , shown is the efficiency to reprogram MEF/Nanog-GFP. MEF/Nanog-GFP cells (1.25 × 10 5 ) were infected with SeVdp vectors, and retroviral vectors were installed with c-Myc / Klf4 / Oct4 / Sox2 as described under “Experimental Procedures.” Then 1.0 × 10 3 of infected cells were seeded onto the feeder cells in 6-well plates and cultured for 14 days ( C ) or for the indicated days ( D ). The number of iPS colonies expressing GFP was determined under fluorescent microscopy. Reprogramming efficiency was indicated as the ratio of the number of EGFP-positive colonies to that of MEF/Nanog-GFP seeded in the well ( C ) or to that of infected MEF/Nanog-GFP seeded in the well ( D ). C , shown is a comparison of reprogramming efficiency with the SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) vector and with retroviral vectors. SeVdp(M/K/O/S) , SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ); RvMX4 , coinfection of ecotropic retroviral vectors installed with c-Myc , Klf4 , Oct4 , and Sox2 separately. D , shown is a comparison of reprogramming efficiency by a single infection of SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) ( SeVdp(M/K/O/S) ) and by coinfections of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ) ( SeVdp(K/O/S ) + SeVdp ( M )). E , characterization of mouse iPS cells generated with SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) is shown. The ES-like colonies emerging from MEF/Nanog-GFP cell lines were fixed, incubated with specific primary antibodies against SeV NP antigen ( left ), Sox2 ( middle ), and SSEA-1 ( right ), then stained with secondary antibodies conjugated with Alexa 555. The cells were then counterstained with DAPI and examined by fluorescence microscopy as described under “Experimental Procedures.” Nanog ( left ), expression of GFP driven by the Nanog promoter. F , gene expression analysis with semiquantitative RT-PCR is shown. Aliquots (2 μg) of total RNA prepared from the cells indicated were analyzed as described under “Experimental Procedures.” Lane 1 , MEF; lane 2 , MEF infected with control vector (SeVdp( Bsr /Δ F / KO )); lane 3 , MEF infected with SeVdp( M/K/O/S ) on day 5 infection; lane 4 , SeVdp-iPS cell clone #2-1; lane 5 , SeVdp-iPS cell clone #9-1; lane 6 , SeVdp-iPS cell clone #13; lane 7 , SeVdp-iPS cell clone #16; lane 8 , SeVdp-iPS cell clone #21; lane 9 , mouse iPS cell generated with retrovirus vectors (RvMX4); lane 10 , mouse ES cell (clone D3). ECAT1 , ES cell-associated transcript 1; FGF4 , fibroblast growth factor 4. G , methylation analysis of Oct4 and Nanog promoters is shown. Methylation profile of CpG in genomic <t>DNA</t> was analyzed by bisulfite sequence analysis as described under “Experimental Procedures.” Open circles , unmethylated cytosine; closed circles , methylated cytosine. The ratio of methylated cytosine is indicated as a percentage of total cytosine residues analyzed. H , a telomerase assay is shown. Telomerase activity in total cell extract prepared from 1 × 10 3 cells was analyzed as described under “Experimental Procedures” and is indicated as the amount of (dTTAGGG) n synthesized. I , histology of teratomas derived from SeVdp-iPS cells is shown. Teratoma formation was studied at 6 weeks after the subcutaneous injection of 1 × 10 6 SeVdp-iPS cells from clone #13 into SCID mice. a–c , low magnification; scale bar = 100 μm. d–h , high magnification observation; scale bar = 20 μm. d , cartilage; e , epidermis; f , hair follicle; g , sweat gland; h , intestinal gland. J , adult chimeras derived from SeVdp-iPS cells (clone #13) are shown. Dark hair indicates donor contribution.
    Template Dna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 97/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/template dna/product/Addgene inc
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    95
    Kaneka Corp dna template
    Reprogramming of MEFs with SeVdp vectors installed with reprogramming genes. A , the genome structure of SeVdp( c-Myc / <t>Klf4</t> / Oct4 / Sox2 ) is shown. B , shown is genome structure of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ), coexisting in a single cell, is shown. C and D , shown is the efficiency to reprogram MEF/Nanog-GFP. MEF/Nanog-GFP cells (1.25 × 10 5 ) were infected with SeVdp vectors, and retroviral vectors were installed with c-Myc / Klf4 / Oct4 / Sox2 as described under “Experimental Procedures.” Then 1.0 × 10 3 of infected cells were seeded onto the feeder cells in 6-well plates and cultured for 14 days ( C ) or for the indicated days ( D ). The number of iPS colonies expressing GFP was determined under fluorescent microscopy. Reprogramming efficiency was indicated as the ratio of the number of EGFP-positive colonies to that of MEF/Nanog-GFP seeded in the well ( C ) or to that of infected MEF/Nanog-GFP seeded in the well ( D ). C , shown is a comparison of reprogramming efficiency with the SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) vector and with retroviral vectors. SeVdp(M/K/O/S) , SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ); RvMX4 , coinfection of ecotropic retroviral vectors installed with c-Myc , Klf4 , Oct4 , and Sox2 separately. D , shown is a comparison of reprogramming efficiency by a single infection of SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) ( SeVdp(M/K/O/S) ) and by coinfections of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ) ( SeVdp(K/O/S ) + SeVdp ( M )). E , characterization of mouse iPS cells generated with SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) is shown. The ES-like colonies emerging from MEF/Nanog-GFP cell lines were fixed, incubated with specific primary antibodies against SeV NP antigen ( left ), Sox2 ( middle ), and SSEA-1 ( right ), then stained with secondary antibodies conjugated with Alexa 555. The cells were then counterstained with DAPI and examined by fluorescence microscopy as described under “Experimental Procedures.” Nanog ( left ), expression of GFP driven by the Nanog promoter. F , gene expression analysis with semiquantitative RT-PCR is shown. Aliquots (2 μg) of total RNA prepared from the cells indicated were analyzed as described under “Experimental Procedures.” Lane 1 , MEF; lane 2 , MEF infected with control vector (SeVdp( Bsr /Δ F / KO )); lane 3 , MEF infected with SeVdp( M/K/O/S ) on day 5 infection; lane 4 , SeVdp-iPS cell clone #2-1; lane 5 , SeVdp-iPS cell clone #9-1; lane 6 , SeVdp-iPS cell clone #13; lane 7 , SeVdp-iPS cell clone #16; lane 8 , SeVdp-iPS cell clone #21; lane 9 , mouse iPS cell generated with retrovirus vectors (RvMX4); lane 10 , mouse ES cell (clone D3). ECAT1 , ES cell-associated transcript 1; FGF4 , fibroblast growth factor 4. G , methylation analysis of Oct4 and Nanog promoters is shown. Methylation profile of CpG in genomic <t>DNA</t> was analyzed by bisulfite sequence analysis as described under “Experimental Procedures.” Open circles , unmethylated cytosine; closed circles , methylated cytosine. The ratio of methylated cytosine is indicated as a percentage of total cytosine residues analyzed. H , a telomerase assay is shown. Telomerase activity in total cell extract prepared from 1 × 10 3 cells was analyzed as described under “Experimental Procedures” and is indicated as the amount of (dTTAGGG) n synthesized. I , histology of teratomas derived from SeVdp-iPS cells is shown. Teratoma formation was studied at 6 weeks after the subcutaneous injection of 1 × 10 6 SeVdp-iPS cells from clone #13 into SCID mice. a–c , low magnification; scale bar = 100 μm. d–h , high magnification observation; scale bar = 20 μm. d , cartilage; e , epidermis; f , hair follicle; g , sweat gland; h , intestinal gland. J , adult chimeras derived from SeVdp-iPS cells (clone #13) are shown. Dark hair indicates donor contribution.
    Dna Template, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 95/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Pharmingen dna template
    Reprogramming of MEFs with SeVdp vectors installed with reprogramming genes. A , the genome structure of SeVdp( c-Myc / <t>Klf4</t> / Oct4 / Sox2 ) is shown. B , shown is genome structure of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ), coexisting in a single cell, is shown. C and D , shown is the efficiency to reprogram MEF/Nanog-GFP. MEF/Nanog-GFP cells (1.25 × 10 5 ) were infected with SeVdp vectors, and retroviral vectors were installed with c-Myc / Klf4 / Oct4 / Sox2 as described under “Experimental Procedures.” Then 1.0 × 10 3 of infected cells were seeded onto the feeder cells in 6-well plates and cultured for 14 days ( C ) or for the indicated days ( D ). The number of iPS colonies expressing GFP was determined under fluorescent microscopy. Reprogramming efficiency was indicated as the ratio of the number of EGFP-positive colonies to that of MEF/Nanog-GFP seeded in the well ( C ) or to that of infected MEF/Nanog-GFP seeded in the well ( D ). C , shown is a comparison of reprogramming efficiency with the SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) vector and with retroviral vectors. SeVdp(M/K/O/S) , SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ); RvMX4 , coinfection of ecotropic retroviral vectors installed with c-Myc , Klf4 , Oct4 , and Sox2 separately. D , shown is a comparison of reprogramming efficiency by a single infection of SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) ( SeVdp(M/K/O/S) ) and by coinfections of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ) ( SeVdp(K/O/S ) + SeVdp ( M )). E , characterization of mouse iPS cells generated with SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) is shown. The ES-like colonies emerging from MEF/Nanog-GFP cell lines were fixed, incubated with specific primary antibodies against SeV NP antigen ( left ), Sox2 ( middle ), and SSEA-1 ( right ), then stained with secondary antibodies conjugated with Alexa 555. The cells were then counterstained with DAPI and examined by fluorescence microscopy as described under “Experimental Procedures.” Nanog ( left ), expression of GFP driven by the Nanog promoter. F , gene expression analysis with semiquantitative RT-PCR is shown. Aliquots (2 μg) of total RNA prepared from the cells indicated were analyzed as described under “Experimental Procedures.” Lane 1 , MEF; lane 2 , MEF infected with control vector (SeVdp( Bsr /Δ F / KO )); lane 3 , MEF infected with SeVdp( M/K/O/S ) on day 5 infection; lane 4 , SeVdp-iPS cell clone #2-1; lane 5 , SeVdp-iPS cell clone #9-1; lane 6 , SeVdp-iPS cell clone #13; lane 7 , SeVdp-iPS cell clone #16; lane 8 , SeVdp-iPS cell clone #21; lane 9 , mouse iPS cell generated with retrovirus vectors (RvMX4); lane 10 , mouse ES cell (clone D3). ECAT1 , ES cell-associated transcript 1; FGF4 , fibroblast growth factor 4. G , methylation analysis of Oct4 and Nanog promoters is shown. Methylation profile of CpG in genomic <t>DNA</t> was analyzed by bisulfite sequence analysis as described under “Experimental Procedures.” Open circles , unmethylated cytosine; closed circles , methylated cytosine. The ratio of methylated cytosine is indicated as a percentage of total cytosine residues analyzed. H , a telomerase assay is shown. Telomerase activity in total cell extract prepared from 1 × 10 3 cells was analyzed as described under “Experimental Procedures” and is indicated as the amount of (dTTAGGG) n synthesized. I , histology of teratomas derived from SeVdp-iPS cells is shown. Teratoma formation was studied at 6 weeks after the subcutaneous injection of 1 × 10 6 SeVdp-iPS cells from clone #13 into SCID mice. a–c , low magnification; scale bar = 100 μm. d–h , high magnification observation; scale bar = 20 μm. d , cartilage; e , epidermis; f , hair follicle; g , sweat gland; h , intestinal gland. J , adult chimeras derived from SeVdp-iPS cells (clone #13) are shown. Dark hair indicates donor contribution.
    Dna Template, supplied by Pharmingen, used in various techniques. Bioz Stars score: 86/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna template/product/Pharmingen
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    dna template - by Bioz Stars, 2020-04
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    Pharmingen dna templates
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    Proposed model for increased expression and activation of DR4 and DR5 following etoposide treatment. Exposure of cells to etoposide causes <t>DNA</t> damage, leading to activation of MEKK1. This activation leads to the phosphorylation and activation of IKKα/β that subsequently phosphorylates and degrades IκB. The degradation of IκB leads to NF-κB-dependent transcription of DR4 and DR5. Increased DR4 and DR5 expression increases TRAIL binding to its receptors. This leads to activation of <t>caspase</t> 8 by its recruitment to the receptor via FADD-like proteins and the subsequent downstream activation of caspase 9. Caspase 8 and caspase 9 activation causes further caspase activation, leading to apoptosis. The antiapoptotic Bcl2 protein inhibits the activation of caspase 9 and prevents etoposide-induced apoptosis.
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    Proposed model for increased expression and activation of DR4 and DR5 following etoposide treatment. Exposure of cells to etoposide causes <t>DNA</t> damage, leading to activation of MEKK1. This activation leads to the phosphorylation and activation of IKKα/β that subsequently phosphorylates and degrades IκB. The degradation of IκB leads to NF-κB-dependent transcription of DR4 and DR5. Increased DR4 and DR5 expression increases TRAIL binding to its receptors. This leads to activation of <t>caspase</t> 8 by its recruitment to the receptor via FADD-like proteins and the subsequent downstream activation of caspase 9. Caspase 8 and caspase 9 activation causes further caspase activation, leading to apoptosis. The antiapoptotic Bcl2 protein inhibits the activation of caspase 9 and prevents etoposide-induced apoptosis.
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    Proposed model for increased expression and activation of DR4 and DR5 following etoposide treatment. Exposure of cells to etoposide causes <t>DNA</t> damage, leading to activation of MEKK1. This activation leads to the phosphorylation and activation of IKKα/β that subsequently phosphorylates and degrades IκB. The degradation of IκB leads to NF-κB-dependent transcription of DR4 and DR5. Increased DR4 and DR5 expression increases TRAIL binding to its receptors. This leads to activation of <t>caspase</t> 8 by its recruitment to the receptor via FADD-like proteins and the subsequent downstream activation of caspase 9. Caspase 8 and caspase 9 activation causes further caspase activation, leading to apoptosis. The antiapoptotic Bcl2 protein inhibits the activation of caspase 9 and prevents etoposide-induced apoptosis.
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    Proposed model for increased expression and activation of DR4 and DR5 following etoposide treatment. Exposure of cells to etoposide causes <t>DNA</t> damage, leading to activation of MEKK1. This activation leads to the phosphorylation and activation of IKKα/β that subsequently phosphorylates and degrades IκB. The degradation of IκB leads to NF-κB-dependent transcription of DR4 and DR5. Increased DR4 and DR5 expression increases TRAIL binding to its receptors. This leads to activation of <t>caspase</t> 8 by its recruitment to the receptor via FADD-like proteins and the subsequent downstream activation of caspase 9. Caspase 8 and caspase 9 activation causes further caspase activation, leading to apoptosis. The antiapoptotic Bcl2 protein inhibits the activation of caspase 9 and prevents etoposide-induced apoptosis.
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    Proposed model for increased expression and activation of DR4 and DR5 following etoposide treatment. Exposure of cells to etoposide causes <t>DNA</t> damage, leading to activation of MEKK1. This activation leads to the phosphorylation and activation of IKKα/β that subsequently phosphorylates and degrades IκB. The degradation of IκB leads to NF-κB-dependent transcription of DR4 and DR5. Increased DR4 and DR5 expression increases TRAIL binding to its receptors. This leads to activation of <t>caspase</t> 8 by its recruitment to the receptor via FADD-like proteins and the subsequent downstream activation of caspase 9. Caspase 8 and caspase 9 activation causes further caspase activation, leading to apoptosis. The antiapoptotic Bcl2 protein inhibits the activation of caspase 9 and prevents etoposide-induced apoptosis.
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    Proposed model for increased expression and activation of DR4 and DR5 following etoposide treatment. Exposure of cells to etoposide causes <t>DNA</t> damage, leading to activation of MEKK1. This activation leads to the phosphorylation and activation of IKKα/β that subsequently phosphorylates and degrades IκB. The degradation of IκB leads to NF-κB-dependent transcription of DR4 and DR5. Increased DR4 and DR5 expression increases TRAIL binding to its receptors. This leads to activation of <t>caspase</t> 8 by its recruitment to the receptor via FADD-like proteins and the subsequent downstream activation of caspase 9. Caspase 8 and caspase 9 activation causes further caspase activation, leading to apoptosis. The antiapoptotic Bcl2 protein inhibits the activation of caspase 9 and prevents etoposide-induced apoptosis.
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    Proposed model for increased expression and activation of DR4 and DR5 following etoposide treatment. Exposure of cells to etoposide causes <t>DNA</t> damage, leading to activation of MEKK1. This activation leads to the phosphorylation and activation of IKKα/β that subsequently phosphorylates and degrades IκB. The degradation of IκB leads to NF-κB-dependent transcription of DR4 and DR5. Increased DR4 and DR5 expression increases TRAIL binding to its receptors. This leads to activation of <t>caspase</t> 8 by its recruitment to the receptor via FADD-like proteins and the subsequent downstream activation of caspase 9. Caspase 8 and caspase 9 activation causes further caspase activation, leading to apoptosis. The antiapoptotic Bcl2 protein inhibits the activation of caspase 9 and prevents etoposide-induced apoptosis.
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    Validation of <t>mRho</t> -specific CRISPRd-mediated gene deletion in vivo A , Schematic of experimental (AAVs-Cas9+GR) and control (AAVs-Cas9+SR) AAV2/8 vector pairs. B , Conventional gene replacement therapy vs CRISPRd plus gene replacement, compound therapy for a heterozygous loci. C , gRNA1 and gRNA2 sequences, their targeting sites on m Rho , and the corresponding sites on hRHO – showing the 12 and 4 mismatches, respectively. D , Representative data of in vitro SpCas9/gRNA cutting. In a cell-free assay, mRho vs hRHO <t>DNA</t> template was mixed with recombinant SpCas9 protein and a single gRNA: gRNA1, gRNA2, or control (scrambled gRNA). (n=3) E , Experimental scheme and timeline for subretinal injection of dual AAV vectors in right eyes of wild-type C57BL/6J mice (using a unique, posterior approach); respecting IACUC protocols, left eyes were uninjected. F , Representative data of Cas9-immunostaining in retinal flat mount. 14 days after Cas9+GR subretinal injection, the retinas were collected and stained for anti-Cas9 antibogy (n=3). Image was assembled from multiple pictures. G , Representative data of PCR analysis of retinal genomic DNA from AAVs-Cas9+GR-injected right eyes and uninjected, fellow (left) eyes. (n=4, two mice data are shown). H , Representative data of mRho gene ablation validated by Sanger sequencing of PCR amplicon from Fig. 2G.
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    Validation of <t>mRho</t> -specific CRISPRd-mediated gene deletion in vivo A , Schematic of experimental (AAVs-Cas9+GR) and control (AAVs-Cas9+SR) AAV2/8 vector pairs. B , Conventional gene replacement therapy vs CRISPRd plus gene replacement, compound therapy for a heterozygous loci. C , gRNA1 and gRNA2 sequences, their targeting sites on m Rho , and the corresponding sites on hRHO – showing the 12 and 4 mismatches, respectively. D , Representative data of in vitro SpCas9/gRNA cutting. In a cell-free assay, mRho vs hRHO <t>DNA</t> template was mixed with recombinant SpCas9 protein and a single gRNA: gRNA1, gRNA2, or control (scrambled gRNA). (n=3) E , Experimental scheme and timeline for subretinal injection of dual AAV vectors in right eyes of wild-type C57BL/6J mice (using a unique, posterior approach); respecting IACUC protocols, left eyes were uninjected. F , Representative data of Cas9-immunostaining in retinal flat mount. 14 days after Cas9+GR subretinal injection, the retinas were collected and stained for anti-Cas9 antibogy (n=3). Image was assembled from multiple pictures. G , Representative data of PCR analysis of retinal genomic DNA from AAVs-Cas9+GR-injected right eyes and uninjected, fellow (left) eyes. (n=4, two mice data are shown). H , Representative data of mRho gene ablation validated by Sanger sequencing of PCR amplicon from Fig. 2G.
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    Image Search Results


    Standard curve demonstrating the range of threshold cycle (Ct) values plotted versus genome equivalents (GE) of Brucella pinnipedialis strain B04-0821 per PCR reaction volume (1.5 µL of DNA). The regression line represents data that were in the linear range. Each point represents the mean value for triplicate runs at each dilution.

    Journal: Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

    Article Title: Application of real-time quantitative PCR assays for detecting marine Brucella spp. in fish

    doi: 10.1177/1040638717733024

    Figure Lengend Snippet: Standard curve demonstrating the range of threshold cycle (Ct) values plotted versus genome equivalents (GE) of Brucella pinnipedialis strain B04-0821 per PCR reaction volume (1.5 µL of DNA). The regression line represents data that were in the linear range. Each point represents the mean value for triplicate runs at each dilution.

    Article Snippet: Total PCR reaction volumes were 15 µL with 4.5 µL of DNA template, 100 µM primers and probe, and a commercial master mix (TaqMan universal master mix II, Life Technologies, Carlsbad, CA).

    Techniques: Polymerase Chain Reaction

    Quantification of maize genomic DNA. Three methods for determining the DNA concentration of the genomic DNA extracts from maize events Bt11, Mon810 and NK603 were compared. DNA extracts were derived from a similar quantity of starting material (40 milligrams as defined by the extraction protocol with multiple extracts combined to exceed the 100 milligrams ERM minimum recommendation) and extracted to the same protocol. Left: NanoDrop spectrophotometer results for each maize event in duplicate (the 260:280 nanometre ratios for all samples ranged from 1.71 to 1.81 and the ratio 260:230 nanometre from 1.69 to 2.59, Bt11 concentration 114.3 nanograms per microlitre); Centre: duplicate loading of DNA from each maize event on an agarose gel (full size gel image shown in Fig. S9 ) compared to ladder of defined DNA concentrations showing intact genomics DNA of large fragment size (Bt11 concentration 52.0 nanograms per microlitre); Right the three quantification methods compared (Qubit determined Bt11 concentration 51.4 nanograms per microlitre).

    Journal: Scientific Reports

    Article Title: Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART)

    doi: 10.1038/s41598-018-36207-4

    Figure Lengend Snippet: Quantification of maize genomic DNA. Three methods for determining the DNA concentration of the genomic DNA extracts from maize events Bt11, Mon810 and NK603 were compared. DNA extracts were derived from a similar quantity of starting material (40 milligrams as defined by the extraction protocol with multiple extracts combined to exceed the 100 milligrams ERM minimum recommendation) and extracted to the same protocol. Left: NanoDrop spectrophotometer results for each maize event in duplicate (the 260:280 nanometre ratios for all samples ranged from 1.71 to 1.81 and the ratio 260:230 nanometre from 1.69 to 2.59, Bt11 concentration 114.3 nanograms per microlitre); Centre: duplicate loading of DNA from each maize event on an agarose gel (full size gel image shown in Fig. S9 ) compared to ladder of defined DNA concentrations showing intact genomics DNA of large fragment size (Bt11 concentration 52.0 nanograms per microlitre); Right the three quantification methods compared (Qubit determined Bt11 concentration 51.4 nanograms per microlitre).

    Article Snippet: Initial quantification of the template DNA was by NanoDrop® spectrophotometer to the manufacturer’s guidelines for double stranded DNA.

    Techniques: Concentration Assay, Derivative Assay, Spectrophotometry, Agarose Gel Electrophoresis

    Effect of the mutated AP-1 sites on the regulation of the TF promoter by PML/RARα. EMSA analysis for the interaction of PML/RARα with the TF promoter was performed using radiolabeled DNA probes spanning from position −250 to −196

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

    Article Title: PML/RAR? fusion protein transactivates the tissue factor promoter through a GAGC-containing element without direct DNA association

    doi: 10.1073/pnas.0915006107

    Figure Lengend Snippet: Effect of the mutated AP-1 sites on the regulation of the TF promoter by PML/RARα. EMSA analysis for the interaction of PML/RARα with the TF promoter was performed using radiolabeled DNA probes spanning from position −250 to −196

    Article Snippet: PML/RARα and RARα proteins were synthesized in vitro with pSG5-RARα and pSG5-PML/RARα as DNA templates according to the manufacturer’s protocol (Promega).

    Techniques:

    RT progression assay determining G-quartet formation in the Sp1 binding region. Cation-dependent pausing of reverse transcription at the guanine-rich elements in the U3 region was analyzed with RNA and DNA templates. A fragment of the RNA/DNA sequence of the Sp1 binding region is shown (top) with G-rich elements (shaded). Strong pauses of the RT near G-rich elements were observed in the presence of 50 mM of KCl, but not LiCl, indicating that these elements are involved in the formation of structure, which is stabilized by potassium ions but destabilized by lithium ions. This is indicative of a G-quadruplex. The cation-independent RT pauses are likely caused by hairpin structures. DNA primer, P; DNA marker, M; KCl, K + ; LiCl, Li + .

    Journal: Biochemistry

    Article Title: U3 Region in the HIV-1 Genome Adopts a G-Quadruplex Structure in Its RNA and DNA Sequence

    doi: 10.1021/bi4016692

    Figure Lengend Snippet: RT progression assay determining G-quartet formation in the Sp1 binding region. Cation-dependent pausing of reverse transcription at the guanine-rich elements in the U3 region was analyzed with RNA and DNA templates. A fragment of the RNA/DNA sequence of the Sp1 binding region is shown (top) with G-rich elements (shaded). Strong pauses of the RT near G-rich elements were observed in the presence of 50 mM of KCl, but not LiCl, indicating that these elements are involved in the formation of structure, which is stabilized by potassium ions but destabilized by lithium ions. This is indicative of a G-quadruplex. The cation-independent RT pauses are likely caused by hairpin structures. DNA primer, P; DNA marker, M; KCl, K + ; LiCl, Li + .

    Article Snippet: Preparation of RNA Templates RNA molecules were transcribed in vitro (Ambion T7-MEGAshortscript kit; Applied Biosystems) from DNA templates amplified by PCR using Vent DNA polymerase (New England BioLabs, Inc.) and two overlapping oligomers with the sequence of the desired region.

    Techniques: Binding Assay, Sequencing, Marker

    Formation of the structure stabilized by potassium ions in the HIV-1 U3 region facilitates RT template switching during reverse transcription. (A) Reconstituted system to analyze the influence of G-rich elements on strand transfer during HIV-1 minus strand DNA synthesis in vitro . Donor and acceptor RNA templates represent two copies of the viral RNA genome; in which reverse transcription is initiated from a 32 P-labeled DNA primer annealed to the donor RNA. The acceptor RNA does not share a homology (circle) with two nucleotides at the 5′ end of the donor RNA. TP, transfer product; DE, donor extension product; and P, DNA primer. (B) A time course of strand transfer reactions performed in the presence of potassium and lithium ions. Samples were collected at 1, 5, 15, and 30 min after the reaction was initiated. Formation of a potassium-dependent structure, anticipated to be a G-quadruplex, in the RNA template paused the RT during minus strand DNA synthesis and influenced the yield of the final products. The transfer efficiency decreased about 37% in reactions with lithium ions, presumably because the templates could not form a G-quadruplex.

    Journal: Biochemistry

    Article Title: U3 Region in the HIV-1 Genome Adopts a G-Quadruplex Structure in Its RNA and DNA Sequence

    doi: 10.1021/bi4016692

    Figure Lengend Snippet: Formation of the structure stabilized by potassium ions in the HIV-1 U3 region facilitates RT template switching during reverse transcription. (A) Reconstituted system to analyze the influence of G-rich elements on strand transfer during HIV-1 minus strand DNA synthesis in vitro . Donor and acceptor RNA templates represent two copies of the viral RNA genome; in which reverse transcription is initiated from a 32 P-labeled DNA primer annealed to the donor RNA. The acceptor RNA does not share a homology (circle) with two nucleotides at the 5′ end of the donor RNA. TP, transfer product; DE, donor extension product; and P, DNA primer. (B) A time course of strand transfer reactions performed in the presence of potassium and lithium ions. Samples were collected at 1, 5, 15, and 30 min after the reaction was initiated. Formation of a potassium-dependent structure, anticipated to be a G-quadruplex, in the RNA template paused the RT during minus strand DNA synthesis and influenced the yield of the final products. The transfer efficiency decreased about 37% in reactions with lithium ions, presumably because the templates could not form a G-quadruplex.

    Article Snippet: Preparation of RNA Templates RNA molecules were transcribed in vitro (Ambion T7-MEGAshortscript kit; Applied Biosystems) from DNA templates amplified by PCR using Vent DNA polymerase (New England BioLabs, Inc.) and two overlapping oligomers with the sequence of the desired region.

    Techniques: DNA Synthesis, In Vitro, Labeling

    CD spectral analysis of the RNA and single stranded DNA with Sp1 binding sites in HIV-1. CD spectra indicate the formation of the parallel G-quadruplex for the RNA template and an antiparallel or hybrid G-quadruplex for single stranded DNA. For reference, a profile of the G-rich sequence (50% of Gs; GGGGGGAUUGUG UGGUACAGUGCAGAGA), which is unable to adopt G-quadruplex structure, is shown in gray.

    Journal: Biochemistry

    Article Title: U3 Region in the HIV-1 Genome Adopts a G-Quadruplex Structure in Its RNA and DNA Sequence

    doi: 10.1021/bi4016692

    Figure Lengend Snippet: CD spectral analysis of the RNA and single stranded DNA with Sp1 binding sites in HIV-1. CD spectra indicate the formation of the parallel G-quadruplex for the RNA template and an antiparallel or hybrid G-quadruplex for single stranded DNA. For reference, a profile of the G-rich sequence (50% of Gs; GGGGGGAUUGUG UGGUACAGUGCAGAGA), which is unable to adopt G-quadruplex structure, is shown in gray.

    Article Snippet: Preparation of RNA Templates RNA molecules were transcribed in vitro (Ambion T7-MEGAshortscript kit; Applied Biosystems) from DNA templates amplified by PCR using Vent DNA polymerase (New England BioLabs, Inc.) and two overlapping oligomers with the sequence of the desired region.

    Techniques: Binding Assay, Sequencing

    PCR amplification of the nuc gene of S. aureus . Lane 1: 100 bp DNA ladder. Lane 5: Amplified PCR product

    Journal: Iranian Journal of Veterinary Research

    Article Title: Isolation, characterization and therapeutic potential assessment of bacteriophages virulent to Staphylococcus aureus associated with goat mastitis

    doi:

    Figure Lengend Snippet: PCR amplification of the nuc gene of S. aureus . Lane 1: 100 bp DNA ladder. Lane 5: Amplified PCR product

    Article Snippet: The PCR mixture consisting of 50 ng template DNA, 20 pmol of each primer and 17 µl PCR master-mix (Qiagen, USA) was diluted up to a 25 µl volume with nuclease free water.

    Techniques: Polymerase Chain Reaction, Amplification

    PCR product in agarose gel electrophoresis. M, Marker DNA λ/ Hind III (M); lane 1 and 2, represented the DNA fragment (2,2 kb) that resulted by PCR using the genome of M. tuberculosis H37RV and INH-resistant M. tuberculosis R2 as templates respectively. The DNA fragment of 2.2 kb corresponds to the katG gene.

    Journal: Journal of Medicine and Life

    Article Title: Molecular Analysis of katG Encoding Catalase-Peroxidase from Clinical Isolate of Isoniazid-Resistant Mycobacterium tuberculosis

    doi:

    Figure Lengend Snippet: PCR product in agarose gel electrophoresis. M, Marker DNA λ/ Hind III (M); lane 1 and 2, represented the DNA fragment (2,2 kb) that resulted by PCR using the genome of M. tuberculosis H37RV and INH-resistant M. tuberculosis R2 as templates respectively. The DNA fragment of 2.2 kb corresponds to the katG gene.

    Article Snippet: The total volume for PCR was 25 µL and it was composed of 5 µL DNA template (25 ng DNA); 20 pmol of each primer; 1.25 units of Taq DNA polymerase (Roche); buffer 1x (10 mM Tris HCl pH 9; 1.5 mM MgCl2 ; 50 mM KCl) and 100 µM of dNTP mix (Roche).

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

    Oncogene sequence analysis. ( top ) Genomic DNA was extracted from BC and electrophoresed in agarose/ethidium bromide. Sample numbers are on top of each lane; mw: molecular weight markers. ( mid ) Exon-intron structure of the TP53 gene. ( bottom ) PCR amplification of the TP53 exons. EX: exon number; multiplex: simultaneous amplification of all exons with optimized primers and amplification procedure; mw: molecular weight markers

    Journal: BMC Cancer

    Article Title: p53, cathepsin D, Bcl-2 are joint prognostic indicators of breast cancer metastatic spreading

    doi: 10.1186/s12885-016-2713-3

    Figure Lengend Snippet: Oncogene sequence analysis. ( top ) Genomic DNA was extracted from BC and electrophoresed in agarose/ethidium bromide. Sample numbers are on top of each lane; mw: molecular weight markers. ( mid ) Exon-intron structure of the TP53 gene. ( bottom ) PCR amplification of the TP53 exons. EX: exon number; multiplex: simultaneous amplification of all exons with optimized primers and amplification procedure; mw: molecular weight markers

    Article Snippet: Reactions were performed in 30 μl total volume (15 μl KapaBlood PCR Kit B, 0.5 μl of template pretreated DNA, 20 pmol primer forward, 20 pmol primer reverse).

    Techniques: Sequencing, Molecular Weight, Polymerase Chain Reaction, Amplification, Multiplex Assay

    Molecular modeling of UTP binding opposite dT template at pol II active site. ( A ) UTP forms a wobble bonding pair with dT template, resulting in the misalignment of its triphosphate group compared to cognate ATP recognition. ( B ) An alternative form of UTP:dT pairing In this form, the UTP shifts toward the minor groove of the RNA/DNA hybrid, leading to steric clash between pol II residues and the incoming nucleotide. The two potential base pairing behaviors were modeled based on the previously reported U:U pair ( 42 ).

    Journal: Nucleic Acids Research

    Article Title: Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution

    doi: 10.1093/nar/gkw220

    Figure Lengend Snippet: Molecular modeling of UTP binding opposite dT template at pol II active site. ( A ) UTP forms a wobble bonding pair with dT template, resulting in the misalignment of its triphosphate group compared to cognate ATP recognition. ( B ) An alternative form of UTP:dT pairing In this form, the UTP shifts toward the minor groove of the RNA/DNA hybrid, leading to steric clash between pol II residues and the incoming nucleotide. The two potential base pairing behaviors were modeled based on the previously reported U:U pair ( 42 ).

    Article Snippet: Briefly, an aliquot of 5′-32 P-labeled RNA (10 μM) was annealed with a 1.5-fold amount of template DNA (15 μM) and 2-fold amount of non-template DNA (20 μM) to form the RNA/DNA scaffold (final stock concentration: 1 μM, defined by RNA concentration) in elongation buffer (20 mM Tris–HCl (pH 7.5), 40 mM KCl, 5 mM MgCl2 ).

    Techniques: Binding Assay

    Dendrogram showing the relatedness of E. coli strains collected from the 72 pigs isolated in four pens as determined by REP-PCR. A condensed dendrogram (using a 60% cut-off value) is shown and contains 20 major clusters. DNA fingerprint similarities were calculated by the curve-based Pearson coefficient, and dendrograms were generated by UPGMA. Dominant profiles previously detected in this farm [ 11 ] are indicated between brackets

    Journal: BMC Microbiology

    Article Title: Genotype variation and genetic relationship among Escherichia coli from nursery pigs located in different pens in the same farm

    doi: 10.1186/s12866-016-0912-3

    Figure Lengend Snippet: Dendrogram showing the relatedness of E. coli strains collected from the 72 pigs isolated in four pens as determined by REP-PCR. A condensed dendrogram (using a 60% cut-off value) is shown and contains 20 major clusters. DNA fingerprint similarities were calculated by the curve-based Pearson coefficient, and dendrograms were generated by UPGMA. Dominant profiles previously detected in this farm [ 11 ] are indicated between brackets

    Article Snippet: The PCR reaction (25 μl) contained 50 ng of template DNA, 3.5 μl of each primer (10 μmol/l stocks), 3.5 μl of Dimethyl Sulfoxide (Sigma-Aldrich) and 13 μl of DreamTaq Green DNA Polymerase (Thermo Scientific, Roskilde, Denmark).

    Techniques: Isolation, Polymerase Chain Reaction, Generated

    Mouse Mbnl3 knockout characterization and Mbnl3 muscle RNA targets in vivo . ( A ) Genomic DNA blot of wild-type ( Mbnl3 + ) and Mbnl3 isoform knockout ( Mbnl3 ΔE2 ), showing the reduction in Kpn I fragment length in Mbnl3 ΔE2 knockouts. ( B ) RT–PCR

    Journal: Human Molecular Genetics

    Article Title: Progressive impairment of muscle regeneration in muscleblind-like 3 isoform knockout mice

    doi: 10.1093/hmg/ddt209

    Figure Lengend Snippet: Mouse Mbnl3 knockout characterization and Mbnl3 muscle RNA targets in vivo . ( A ) Genomic DNA blot of wild-type ( Mbnl3 + ) and Mbnl3 isoform knockout ( Mbnl3 ΔE2 ), showing the reduction in Kpn I fragment length in Mbnl3 ΔE2 knockouts. ( B ) RT–PCR

    Article Snippet: Hybridization probes were generated by [α-32 P]dCTP-labeling using 50 ng of DNA template (Mbnl1, Mbnl3, Ppia) with DNA Labeling Beads-dCTP (GE Healthcare) and purified with Illustra ProbeQuant G-50 Micro Columns (GE Healthcare).

    Techniques: Knock-Out, In Vivo, Reverse Transcription Polymerase Chain Reaction

    Inverse PCR (iPCR) and sequencing of the boundary region surrounding the Mk + 1.3 locus. ( a ) Agarose gel electrophoresis of nested PCR products stained with ethidium bromide. The gel is the full length gel and lanes have not been cropped or stitched. ( b ) Chromatograms generated from direct sequencing of the iPCR products. Black and red letters above the chromatograms indicate the sequences of chromosomal positions chr 7: 62,416,891–62,416,891 and chr 7: 62,268,615–62,268,638 (on the University of California, Santa Cruz, genome browser (GRCm38/mm10)), respectively. W: wild-type DNA, Tg: hemizygous transgenic DNA, M: molecular size marker.

    Journal: Scientific Reports

    Article Title: Application of droplet digital PCR in the analysis of genome integration and organization of the transgene in BAC transgenic mice

    doi: 10.1038/s41598-018-25001-x

    Figure Lengend Snippet: Inverse PCR (iPCR) and sequencing of the boundary region surrounding the Mk + 1.3 locus. ( a ) Agarose gel electrophoresis of nested PCR products stained with ethidium bromide. The gel is the full length gel and lanes have not been cropped or stitched. ( b ) Chromatograms generated from direct sequencing of the iPCR products. Black and red letters above the chromatograms indicate the sequences of chromosomal positions chr 7: 62,416,891–62,416,891 and chr 7: 62,268,615–62,268,638 (on the University of California, Santa Cruz, genome browser (GRCm38/mm10)), respectively. W: wild-type DNA, Tg: hemizygous transgenic DNA, M: molecular size marker.

    Article Snippet: Nested amplification around the circular DNA using primers oriented in a direction opposite to that of conventional PCR was performed using reaction mixtures containing DNA template, 1 × Ex Taq buffer (containing 2.0 mM of MgCl2 ), 250 μM of dNTP mix, 0.5 μM of each primer, and 0.25 U/μl of Ex Taq DNA Polymerase (Takara Bio, Otsu, Japan).

    Techniques: Inverse PCR, Sequencing, Agarose Gel Electrophoresis, Nested PCR, Staining, Generated, Transgenic Assay, Marker

    Procedure for the AAV Barcode-Seq analysis. We applied DNA-barcoded AAV library stocks to tissue culture cells for receptor binding and in vitro transduction assays. We injected them into mice to investigate blood clearance rate, tissue tropism, reactivity to neutralizing antibodies and anti-AAV neutralizing antibody epitope mapping. We then collected various samples, extracted DNA, PCR-amplified AAV clone-specific Virus Bar Codes (VBCs) using Sample-specific Bar Code (SBC)-attached PCR primers. We mixed all the VBC–PCR amplicons in a pool, and sequenced them on the Illumina platform. We then converted raw sequence read number data to PD values by a computational algorithm.

    Journal: Nature Communications

    Article Title: Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing

    doi: 10.1038/ncomms4075

    Figure Lengend Snippet: Procedure for the AAV Barcode-Seq analysis. We applied DNA-barcoded AAV library stocks to tissue culture cells for receptor binding and in vitro transduction assays. We injected them into mice to investigate blood clearance rate, tissue tropism, reactivity to neutralizing antibodies and anti-AAV neutralizing antibody epitope mapping. We then collected various samples, extracted DNA, PCR-amplified AAV clone-specific Virus Bar Codes (VBCs) using Sample-specific Bar Code (SBC)-attached PCR primers. We mixed all the VBC–PCR amplicons in a pool, and sequenced them on the Illumina platform. We then converted raw sequence read number data to PD values by a computational algorithm.

    Article Snippet: Over a dynamic range of 104 , the relative abundance of DNA templates and Illumina sequencing read numbers in a sample were positively correlated with Pearson’s correlation coefficients of 0.93 (lt-VBCs) and 0.97 (rt-VBCs) on average ( ).

    Techniques: Binding Assay, In Vitro, Transduction, Injection, Mouse Assay, Polymerase Chain Reaction, Amplification, Sequencing

    Reprogramming of MEFs with SeVdp vectors installed with reprogramming genes. A , the genome structure of SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) is shown. B , shown is genome structure of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ), coexisting in a single cell, is shown. C and D , shown is the efficiency to reprogram MEF/Nanog-GFP. MEF/Nanog-GFP cells (1.25 × 10 5 ) were infected with SeVdp vectors, and retroviral vectors were installed with c-Myc / Klf4 / Oct4 / Sox2 as described under “Experimental Procedures.” Then 1.0 × 10 3 of infected cells were seeded onto the feeder cells in 6-well plates and cultured for 14 days ( C ) or for the indicated days ( D ). The number of iPS colonies expressing GFP was determined under fluorescent microscopy. Reprogramming efficiency was indicated as the ratio of the number of EGFP-positive colonies to that of MEF/Nanog-GFP seeded in the well ( C ) or to that of infected MEF/Nanog-GFP seeded in the well ( D ). C , shown is a comparison of reprogramming efficiency with the SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) vector and with retroviral vectors. SeVdp(M/K/O/S) , SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ); RvMX4 , coinfection of ecotropic retroviral vectors installed with c-Myc , Klf4 , Oct4 , and Sox2 separately. D , shown is a comparison of reprogramming efficiency by a single infection of SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) ( SeVdp(M/K/O/S) ) and by coinfections of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ) ( SeVdp(K/O/S ) + SeVdp ( M )). E , characterization of mouse iPS cells generated with SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) is shown. The ES-like colonies emerging from MEF/Nanog-GFP cell lines were fixed, incubated with specific primary antibodies against SeV NP antigen ( left ), Sox2 ( middle ), and SSEA-1 ( right ), then stained with secondary antibodies conjugated with Alexa 555. The cells were then counterstained with DAPI and examined by fluorescence microscopy as described under “Experimental Procedures.” Nanog ( left ), expression of GFP driven by the Nanog promoter. F , gene expression analysis with semiquantitative RT-PCR is shown. Aliquots (2 μg) of total RNA prepared from the cells indicated were analyzed as described under “Experimental Procedures.” Lane 1 , MEF; lane 2 , MEF infected with control vector (SeVdp( Bsr /Δ F / KO )); lane 3 , MEF infected with SeVdp( M/K/O/S ) on day 5 infection; lane 4 , SeVdp-iPS cell clone #2-1; lane 5 , SeVdp-iPS cell clone #9-1; lane 6 , SeVdp-iPS cell clone #13; lane 7 , SeVdp-iPS cell clone #16; lane 8 , SeVdp-iPS cell clone #21; lane 9 , mouse iPS cell generated with retrovirus vectors (RvMX4); lane 10 , mouse ES cell (clone D3). ECAT1 , ES cell-associated transcript 1; FGF4 , fibroblast growth factor 4. G , methylation analysis of Oct4 and Nanog promoters is shown. Methylation profile of CpG in genomic DNA was analyzed by bisulfite sequence analysis as described under “Experimental Procedures.” Open circles , unmethylated cytosine; closed circles , methylated cytosine. The ratio of methylated cytosine is indicated as a percentage of total cytosine residues analyzed. H , a telomerase assay is shown. Telomerase activity in total cell extract prepared from 1 × 10 3 cells was analyzed as described under “Experimental Procedures” and is indicated as the amount of (dTTAGGG) n synthesized. I , histology of teratomas derived from SeVdp-iPS cells is shown. Teratoma formation was studied at 6 weeks after the subcutaneous injection of 1 × 10 6 SeVdp-iPS cells from clone #13 into SCID mice. a–c , low magnification; scale bar = 100 μm. d–h , high magnification observation; scale bar = 20 μm. d , cartilage; e , epidermis; f , hair follicle; g , sweat gland; h , intestinal gland. J , adult chimeras derived from SeVdp-iPS cells (clone #13) are shown. Dark hair indicates donor contribution.

    Journal: The Journal of Biological Chemistry

    Article Title: Development of Defective and Persistent Sendai Virus Vector

    doi: 10.1074/jbc.M110.183780

    Figure Lengend Snippet: Reprogramming of MEFs with SeVdp vectors installed with reprogramming genes. A , the genome structure of SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) is shown. B , shown is genome structure of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ), coexisting in a single cell, is shown. C and D , shown is the efficiency to reprogram MEF/Nanog-GFP. MEF/Nanog-GFP cells (1.25 × 10 5 ) were infected with SeVdp vectors, and retroviral vectors were installed with c-Myc / Klf4 / Oct4 / Sox2 as described under “Experimental Procedures.” Then 1.0 × 10 3 of infected cells were seeded onto the feeder cells in 6-well plates and cultured for 14 days ( C ) or for the indicated days ( D ). The number of iPS colonies expressing GFP was determined under fluorescent microscopy. Reprogramming efficiency was indicated as the ratio of the number of EGFP-positive colonies to that of MEF/Nanog-GFP seeded in the well ( C ) or to that of infected MEF/Nanog-GFP seeded in the well ( D ). C , shown is a comparison of reprogramming efficiency with the SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) vector and with retroviral vectors. SeVdp(M/K/O/S) , SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ); RvMX4 , coinfection of ecotropic retroviral vectors installed with c-Myc , Klf4 , Oct4 , and Sox2 separately. D , shown is a comparison of reprogramming efficiency by a single infection of SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) ( SeVdp(M/K/O/S) ) and by coinfections of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ) ( SeVdp(K/O/S ) + SeVdp ( M )). E , characterization of mouse iPS cells generated with SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) is shown. The ES-like colonies emerging from MEF/Nanog-GFP cell lines were fixed, incubated with specific primary antibodies against SeV NP antigen ( left ), Sox2 ( middle ), and SSEA-1 ( right ), then stained with secondary antibodies conjugated with Alexa 555. The cells were then counterstained with DAPI and examined by fluorescence microscopy as described under “Experimental Procedures.” Nanog ( left ), expression of GFP driven by the Nanog promoter. F , gene expression analysis with semiquantitative RT-PCR is shown. Aliquots (2 μg) of total RNA prepared from the cells indicated were analyzed as described under “Experimental Procedures.” Lane 1 , MEF; lane 2 , MEF infected with control vector (SeVdp( Bsr /Δ F / KO )); lane 3 , MEF infected with SeVdp( M/K/O/S ) on day 5 infection; lane 4 , SeVdp-iPS cell clone #2-1; lane 5 , SeVdp-iPS cell clone #9-1; lane 6 , SeVdp-iPS cell clone #13; lane 7 , SeVdp-iPS cell clone #16; lane 8 , SeVdp-iPS cell clone #21; lane 9 , mouse iPS cell generated with retrovirus vectors (RvMX4); lane 10 , mouse ES cell (clone D3). ECAT1 , ES cell-associated transcript 1; FGF4 , fibroblast growth factor 4. G , methylation analysis of Oct4 and Nanog promoters is shown. Methylation profile of CpG in genomic DNA was analyzed by bisulfite sequence analysis as described under “Experimental Procedures.” Open circles , unmethylated cytosine; closed circles , methylated cytosine. The ratio of methylated cytosine is indicated as a percentage of total cytosine residues analyzed. H , a telomerase assay is shown. Telomerase activity in total cell extract prepared from 1 × 10 3 cells was analyzed as described under “Experimental Procedures” and is indicated as the amount of (dTTAGGG) n synthesized. I , histology of teratomas derived from SeVdp-iPS cells is shown. Teratoma formation was studied at 6 weeks after the subcutaneous injection of 1 × 10 6 SeVdp-iPS cells from clone #13 into SCID mice. a–c , low magnification; scale bar = 100 μm. d–h , high magnification observation; scale bar = 20 μm. d , cartilage; e , epidermis; f , hair follicle; g , sweat gland; h , intestinal gland. J , adult chimeras derived from SeVdp-iPS cells (clone #13) are shown. Dark hair indicates donor contribution.

    Article Snippet: Retroviral vectors installed separately with Oct3/4 , Sox2 , Klf4 , and c-Myc (RvMX4) were prepared as described ( ) using template DNA obtained from Addgene (Cambridge, MA).

    Techniques: Infection, Cell Culture, Expressing, Microscopy, Plasmid Preparation, Generated, Incubation, Staining, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Methylation, Sequencing, Telomerase Assay, Activity Assay, Synthesized, Derivative Assay, Injection, Mouse Assay

    MEG3 RNA 3D structure models. ( A ) MEG3 RNA 3D structure (helix-ladder) predicted from natively folded, in vitro transcribed RNA secondary structure. Conserved motifs I–V are shown in red, orange, blue, green, and brown, respectively. Phosphate atoms of nucleotides predicted to interact with the PRC2 complex by CLIP (blue) or SHAPE-MaP (gray) are depicted as semi-transparent spheres scaled to 4-times the expected van der Waals radii to increase visibility. The phosphates in M-I nucleotides previously found to interact with DNA duplex are also shown (red spheres). The PRC2 surface representation was generated from cryo-EM model structure PDB coordinates (PMID: 29348366; PDB accession number 6c24). The EZH2 and SUZ12 subunits are highlighted (lime green, sky blue) together with elements of those subunits implicated in RNA binding. Specifically, RNA-binding EZH2 residues 342–368 are housed within a larger segment (residues 307–422) that is unresolved in the model structure and approximated here by a 25 Å radius, semitransparent forest green sphere. The ‘foot’ region of SUZ12, depicted here in dark blue, is also implicated in RNA binding, perhaps to the extreme 3′ terminus of M-I. ( B ) Surface representation of native, in vitro transcribed (left) and nuclear ex-vivo (right) MEG3 RNAs. Color coding for motifs I–V matches that in (A).

    Journal: Nucleic Acids Research

    Article Title: Structural characterization of maternally expressed gene 3 RNA reveals conserved motifs and potential sites of interaction with polycomb repressive complex 2

    doi: 10.1093/nar/gky722

    Figure Lengend Snippet: MEG3 RNA 3D structure models. ( A ) MEG3 RNA 3D structure (helix-ladder) predicted from natively folded, in vitro transcribed RNA secondary structure. Conserved motifs I–V are shown in red, orange, blue, green, and brown, respectively. Phosphate atoms of nucleotides predicted to interact with the PRC2 complex by CLIP (blue) or SHAPE-MaP (gray) are depicted as semi-transparent spheres scaled to 4-times the expected van der Waals radii to increase visibility. The phosphates in M-I nucleotides previously found to interact with DNA duplex are also shown (red spheres). The PRC2 surface representation was generated from cryo-EM model structure PDB coordinates (PMID: 29348366; PDB accession number 6c24). The EZH2 and SUZ12 subunits are highlighted (lime green, sky blue) together with elements of those subunits implicated in RNA binding. Specifically, RNA-binding EZH2 residues 342–368 are housed within a larger segment (residues 307–422) that is unresolved in the model structure and approximated here by a 25 Å radius, semitransparent forest green sphere. The ‘foot’ region of SUZ12, depicted here in dark blue, is also implicated in RNA binding, perhaps to the extreme 3′ terminus of M-I. ( B ) Surface representation of native, in vitro transcribed (left) and nuclear ex-vivo (right) MEG3 RNAs. Color coding for motifs I–V matches that in (A).

    Article Snippet: In vitro transcription of full length sense and antisense Meg3 RNA and its non-denaturing purification The plasmid (pCI-flMeg3) used to generate the DNA template for in vitro transcription was created by cloning full length Meg3 DNA between XhoI and NotI sites of pCI mammalian expression vector (Addgene).

    Techniques: In Vitro, Cross-linking Immunoprecipitation, Generated, RNA Binding Assay, Ex Vivo

    Proposed model for increased expression and activation of DR4 and DR5 following etoposide treatment. Exposure of cells to etoposide causes DNA damage, leading to activation of MEKK1. This activation leads to the phosphorylation and activation of IKKα/β that subsequently phosphorylates and degrades IκB. The degradation of IκB leads to NF-κB-dependent transcription of DR4 and DR5. Increased DR4 and DR5 expression increases TRAIL binding to its receptors. This leads to activation of caspase 8 by its recruitment to the receptor via FADD-like proteins and the subsequent downstream activation of caspase 9. Caspase 8 and caspase 9 activation causes further caspase activation, leading to apoptosis. The antiapoptotic Bcl2 protein inhibits the activation of caspase 9 and prevents etoposide-induced apoptosis.

    Journal: Molecular and Cellular Biology

    Article Title: Increased Expression of Death Receptors 4 and 5 Synergizes the Apoptosis Response to Combined Treatment with Etoposide and TRAIL

    doi:

    Figure Lengend Snippet: Proposed model for increased expression and activation of DR4 and DR5 following etoposide treatment. Exposure of cells to etoposide causes DNA damage, leading to activation of MEKK1. This activation leads to the phosphorylation and activation of IKKα/β that subsequently phosphorylates and degrades IκB. The degradation of IκB leads to NF-κB-dependent transcription of DR4 and DR5. Increased DR4 and DR5 expression increases TRAIL binding to its receptors. This leads to activation of caspase 8 by its recruitment to the receptor via FADD-like proteins and the subsequent downstream activation of caspase 9. Caspase 8 and caspase 9 activation causes further caspase activation, leading to apoptosis. The antiapoptotic Bcl2 protein inhibits the activation of caspase 9 and prevents etoposide-induced apoptosis.

    Article Snippet: An hAPO3c probe set containing DNA templates for caspase 8, FASL, FAS, DR3, decoy receptor 1 (DcR1), DR4, DR5, TRAIL, TNFR p55, TRADD, receptor interacting protein (RIP), L32, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or an hSTRESS probe set containing DNA templates for Bcl-x, Bcl2, Bax, p21, GADD45, p53, Mcl1, L32, and GAPDH (PharMingen) was used for T7 RNA-polymerase direct synthesis of [α32 -P]UTP-labeled antisense RNA probes.

    Techniques: Expressing, Activation Assay, Binding Assay

    Validation of mRho -specific CRISPRd-mediated gene deletion in vivo A , Schematic of experimental (AAVs-Cas9+GR) and control (AAVs-Cas9+SR) AAV2/8 vector pairs. B , Conventional gene replacement therapy vs CRISPRd plus gene replacement, compound therapy for a heterozygous loci. C , gRNA1 and gRNA2 sequences, their targeting sites on m Rho , and the corresponding sites on hRHO – showing the 12 and 4 mismatches, respectively. D , Representative data of in vitro SpCas9/gRNA cutting. In a cell-free assay, mRho vs hRHO DNA template was mixed with recombinant SpCas9 protein and a single gRNA: gRNA1, gRNA2, or control (scrambled gRNA). (n=3) E , Experimental scheme and timeline for subretinal injection of dual AAV vectors in right eyes of wild-type C57BL/6J mice (using a unique, posterior approach); respecting IACUC protocols, left eyes were uninjected. F , Representative data of Cas9-immunostaining in retinal flat mount. 14 days after Cas9+GR subretinal injection, the retinas were collected and stained for anti-Cas9 antibogy (n=3). Image was assembled from multiple pictures. G , Representative data of PCR analysis of retinal genomic DNA from AAVs-Cas9+GR-injected right eyes and uninjected, fellow (left) eyes. (n=4, two mice data are shown). H , Representative data of mRho gene ablation validated by Sanger sequencing of PCR amplicon from Fig. 2G.

    Journal: Ophthalmology

    Article Title: CRISPR-based genome surgery for the treatment of autosomal dominant retinitis pigmentosa

    doi: 10.1016/j.ophtha.2018.04.001

    Figure Lengend Snippet: Validation of mRho -specific CRISPRd-mediated gene deletion in vivo A , Schematic of experimental (AAVs-Cas9+GR) and control (AAVs-Cas9+SR) AAV2/8 vector pairs. B , Conventional gene replacement therapy vs CRISPRd plus gene replacement, compound therapy for a heterozygous loci. C , gRNA1 and gRNA2 sequences, their targeting sites on m Rho , and the corresponding sites on hRHO – showing the 12 and 4 mismatches, respectively. D , Representative data of in vitro SpCas9/gRNA cutting. In a cell-free assay, mRho vs hRHO DNA template was mixed with recombinant SpCas9 protein and a single gRNA: gRNA1, gRNA2, or control (scrambled gRNA). (n=3) E , Experimental scheme and timeline for subretinal injection of dual AAV vectors in right eyes of wild-type C57BL/6J mice (using a unique, posterior approach); respecting IACUC protocols, left eyes were uninjected. F , Representative data of Cas9-immunostaining in retinal flat mount. 14 days after Cas9+GR subretinal injection, the retinas were collected and stained for anti-Cas9 antibogy (n=3). Image was assembled from multiple pictures. G , Representative data of PCR analysis of retinal genomic DNA from AAVs-Cas9+GR-injected right eyes and uninjected, fellow (left) eyes. (n=4, two mice data are shown). H , Representative data of mRho gene ablation validated by Sanger sequencing of PCR amplicon from Fig. 2G.

    Article Snippet: To validate the targeting efficiency of our system, gRNA (25 ng/µl) was added to the reaction mixture alongside Cas9 protein (30 ng/µl, NEB) and template mRho DNA (20 ng/µl, 750 bp) covering both targeting sites of gRNA1 and gRNA2, and they were subsequently incubated at 37° C for 2 hr.

    Techniques: In Vivo, Plasmid Preparation, In Vitro, Cell-Free Assay, Recombinant, Injection, Mouse Assay, Immunostaining, Staining, Polymerase Chain Reaction, Sequencing, Amplification