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TaKaRa system kit
System Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/system kit/product/TaKaRa
Average 93 stars, based on 2 article reviews
Price from $9.99 to $1999.99
system kit - by Bioz Stars, 2020-04
93/100 stars

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Related Articles

Transduction:

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: Paragraph title: Cloning and retroviral transduction of mutant and WT HER2 into cell lines ... These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA).

Clone Assay:

Article Title: Analysis of somatic mutations across the kinome reveals loss-of-function mutations in multiple cancer types
Article Snippet: .. Constructs were then shuttled into the pCFG5 retroviral vector (which includes a zeocin resistance marker and IRES-GFP sequence) using the In-Fusion HD cloning system kit (Clonetech), and verified by full-length Sanger sequencing. .. For KDR, TGFBR1 and CHEK2, a c-terminal FLAG tag was introduced.

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: .. These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. The desired constructs were verified using Sanger sequencing.

Flow Cytometry:

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. Cell lines were subjected to 1-2 weeks of zeocin selection and transgene expression was verified by flow cytometry analysis for GFP expression (always > 95%, data not shown) and western blot analysis for HER2.

Selection:

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. Cell lines were subjected to 1-2 weeks of zeocin selection and transgene expression was verified by flow cytometry analysis for GFP expression (always > 95%, data not shown) and western blot analysis for HER2.

Mutagenesis:

Article Title: Analysis of somatic mutations across the kinome reveals loss-of-function mutations in multiple cancer types
Article Snippet: Mutations were introduced using QuikChange II site-directed mutagenesis (Agilent). .. Constructs were then shuttled into the pCFG5 retroviral vector (which includes a zeocin resistance marker and IRES-GFP sequence) using the In-Fusion HD cloning system kit (Clonetech), and verified by full-length Sanger sequencing.

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: Paragraph title: Cloning and retroviral transduction of mutant and WT HER2 into cell lines ... These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA).

Marker:

Article Title: Analysis of somatic mutations across the kinome reveals loss-of-function mutations in multiple cancer types
Article Snippet: .. Constructs were then shuttled into the pCFG5 retroviral vector (which includes a zeocin resistance marker and IRES-GFP sequence) using the In-Fusion HD cloning system kit (Clonetech), and verified by full-length Sanger sequencing. .. For KDR, TGFBR1 and CHEK2, a c-terminal FLAG tag was introduced.

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: .. These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. The desired constructs were verified using Sanger sequencing.

Cytometry:

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. Cell lines were subjected to 1-2 weeks of zeocin selection and transgene expression was verified by flow cytometry analysis for GFP expression (always > 95%, data not shown) and western blot analysis for HER2.

Construct:

Article Title: Analysis of somatic mutations across the kinome reveals loss-of-function mutations in multiple cancer types
Article Snippet: .. Constructs were then shuttled into the pCFG5 retroviral vector (which includes a zeocin resistance marker and IRES-GFP sequence) using the In-Fusion HD cloning system kit (Clonetech), and verified by full-length Sanger sequencing. .. For KDR, TGFBR1 and CHEK2, a c-terminal FLAG tag was introduced.

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: .. These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. The desired constructs were verified using Sanger sequencing.

Produced:

Article Title: Analysis of somatic mutations across the kinome reveals loss-of-function mutations in multiple cancer types
Article Snippet: Constructs were then shuttled into the pCFG5 retroviral vector (which includes a zeocin resistance marker and IRES-GFP sequence) using the In-Fusion HD cloning system kit (Clonetech), and verified by full-length Sanger sequencing. .. For ERBB2, TGFBR1 and KDR, retroviral particles were produced using ϕNX amphotrophic packaging cells.

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. Retroviral particles were produced using φNX amphotrophic packaging cell line and cell lines were infected as per prior publications ( , , ).

FLAG-tag:

Article Title: Analysis of somatic mutations across the kinome reveals loss-of-function mutations in multiple cancer types
Article Snippet: Constructs were then shuttled into the pCFG5 retroviral vector (which includes a zeocin resistance marker and IRES-GFP sequence) using the In-Fusion HD cloning system kit (Clonetech), and verified by full-length Sanger sequencing. .. For KDR, TGFBR1 and CHEK2, a c-terminal FLAG tag was introduced.

Infection:

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. Retroviral particles were produced using φNX amphotrophic packaging cell line and cell lines were infected as per prior publications ( , , ).

Expressing:

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. Cell lines were subjected to 1-2 weeks of zeocin selection and transgene expression was verified by flow cytometry analysis for GFP expression (always > 95%, data not shown) and western blot analysis for HER2.

Sequencing:

Article Title: Analysis of somatic mutations across the kinome reveals loss-of-function mutations in multiple cancer types
Article Snippet: .. Constructs were then shuttled into the pCFG5 retroviral vector (which includes a zeocin resistance marker and IRES-GFP sequence) using the In-Fusion HD cloning system kit (Clonetech), and verified by full-length Sanger sequencing. .. For KDR, TGFBR1 and CHEK2, a c-terminal FLAG tag was introduced.

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: .. These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. The desired constructs were verified using Sanger sequencing.

Western Blot:

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. Cell lines were subjected to 1-2 weeks of zeocin selection and transgene expression was verified by flow cytometry analysis for GFP expression (always > 95%, data not shown) and western blot analysis for HER2.

Plasmid Preparation:

Article Title: Analysis of somatic mutations across the kinome reveals loss-of-function mutations in multiple cancer types
Article Snippet: .. Constructs were then shuttled into the pCFG5 retroviral vector (which includes a zeocin resistance marker and IRES-GFP sequence) using the In-Fusion HD cloning system kit (Clonetech), and verified by full-length Sanger sequencing. .. For KDR, TGFBR1 and CHEK2, a c-terminal FLAG tag was introduced.

Article Title: Activating HER2 mutations in HER2 gene amplification negative breast cancer
Article Snippet: .. These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit (Clontech, Mountain View, CA). .. The desired constructs were verified using Sanger sequencing.

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  • 92
    TaKaRa takara clontech ribogone kits
    Properties of the rRNA depleted libraries: a ) Fraction of reads mapping to nuclear rRNA shown. Site number indicated by color. Intact samples are shown as circles, degraded samples are shown as diamonds. Kit abbreviations: RZ = RiboZero Gold, LX = Lexogen RiboCop, Q = Qiagen GeneRead rRNA Depletion, NE = NEBNext rRNA Depletion, K=Kapa RiboErase, CR = <t>Clontech</t> <t>Ribogone,</t> CZ = SMARTer Pico total RNA. b ) Reads were mapped to exons in UCSC known gene and scored based on strand of alignment. c ) Fraction of reads mapping to mt rRNA shown as in A. *- RiboZero site 3 used standard RiboZero instead of RiboZero Gold
    Takara Clontech Ribogone Kits, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/takara clontech ribogone kits/product/TaKaRa
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    takara clontech ribogone kits - by Bioz Stars, 2020-04
    92/100 stars
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    85
    TaKaRa cdna preparation kit
    Northern analyses of a few select forward subtracted SSH <t>cDNA</t> clones . RNA isolated from the leaves of the control and 425 mM NaCl-treated plants and blotted onto Hybond N + membrane was hybridized with the individual radiolabelled ESTs. A RNA blot each for the control and NaCl-treated sample was hybridized with <t>PCR</t> amplified radiolabelled actin fragment. The horizontal bars against the individual genes represent increase (in%) in transcripts of the respective genes in response to NaCl treatment of the plant when compared to control. The values were obtained after normalization of the blot intensities of actin for the control and NaCl treated sample.
    Cdna Preparation Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdna preparation kit/product/TaKaRa
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cdna preparation kit - by Bioz Stars, 2020-04
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    93
    TaKaRa mrna purification kit
    The susceptible phenotype of selected mutant lines . (A) Three-week-old NB, pish mutant lines, NC8589 andND2043, and ttm mutant lines were inoculated with M. oryzae containing avrPish . The photographs were taken 7 days post inoculation. (B) Expression of the Pish gene. Total <t>RNA</t> was extracted from leaf tissues of NB and each mutant. Samples (1 μg) of poly(A) + RNA were separated by gel electrophoresis, blotted, and hybridized with radiolabeled probes as indicated. The Actin probe was used as a loading control. The closed arrowhead indicates the size of the Pish <t>mRNA.</t> The asterisk indicates a non-specific signal detected by the Pish probe.
    Mrna Purification Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna purification kit/product/TaKaRa
    Average 93 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    mrna purification kit - by Bioz Stars, 2020-04
    93/100 stars
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    96
    TaKaRa resolvase based mutation detection kit
    (A) AAV vector maps depicting the AAV-P Mecp2 -Cas9 vector and an inducible gRNA vector, AAV-gRNAi-TetR-GFP. (B) The gRNAi plasmid described in (A) , containing either a Tet gRNA or no gRNA (Empty), were co-transfected with pX330 Empty into N2A cells in the presence or absence of Dox. Nighty six hours post transfection, the cells were harvested, the genomic DNA was isolated and PCR, and RFLP analysis was performed to assess if genome editing had occurred at the Tet2 locus. Genome editing was not observed in cells that had received the gRNA Empty plasmid either in the presence or absence of Dox as expected. Cells that were transfected with a plasmid containing the gRNA Tet2 plasmid, did exhibit editing in the presence of Dox, but not in the absence of Dox, indicating that genome editing could be regulated in a Dox dependent manner. Similar results were observed in at least 3 independent samples per group. (C) The H1/TO promoter gRNA expression cassette was compared to gRNA expression cassettes composed of either a full length H1/TO promoter (H1-L/TO), a U6/TO promoter or a non-inducible U6 promoter (U6) for their ability to edit the Tet2 locus. Cells were transfected with the respective vectors in the presence or absence of Dox and genome editing was assessed 96 h post transfection ( n = 4 per groups). (D) Quantification of genome editing revealed that the inducible promoter gRNA expression cassettes were not significantly different from each other (n.s. = p ≤ 0.0778) in their ability to edit the Tet2 locus and they were all slightly but significantly different from the non-inducible U6 promoter construct ( * p ≤ 0.003). Four independent samples were screened with similar results. (E) Partial sequences of the inducible promoters used, highlighting the tetracycline operators (Tet-O) and transcriptional start site. (F) Genomic DNA from the same U6, H1/TO, and UTC samples used for (C,D) were subjected to a mutation detection kit (Clontech), in which the PCR amplified product was denatured, annealed and digested with <t>Resolvase</t> to directly detect edited DNA. (G) Levels of editing were comparable to those seen using the restriction digest PCR/RFLP analysis used for (C,D) . Again, levels of editing were significantly higher in the U6 group, compared to the H1/TO group (# p ≤ 0.005, Two Tailed T -test).
    Resolvase Based Mutation Detection Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/resolvase based mutation detection kit/product/TaKaRa
    Average 96 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    resolvase based mutation detection kit - by Bioz Stars, 2020-04
    96/100 stars
      Buy from Supplier

    Image Search Results


    Properties of the rRNA depleted libraries: a ) Fraction of reads mapping to nuclear rRNA shown. Site number indicated by color. Intact samples are shown as circles, degraded samples are shown as diamonds. Kit abbreviations: RZ = RiboZero Gold, LX = Lexogen RiboCop, Q = Qiagen GeneRead rRNA Depletion, NE = NEBNext rRNA Depletion, K=Kapa RiboErase, CR = Clontech Ribogone, CZ = SMARTer Pico total RNA. b ) Reads were mapped to exons in UCSC known gene and scored based on strand of alignment. c ) Fraction of reads mapping to mt rRNA shown as in A. *- RiboZero site 3 used standard RiboZero instead of RiboZero Gold

    Journal: BMC Genomics

    Article Title: Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction

    doi: 10.1186/s12864-018-4585-1

    Figure Lengend Snippet: Properties of the rRNA depleted libraries: a ) Fraction of reads mapping to nuclear rRNA shown. Site number indicated by color. Intact samples are shown as circles, degraded samples are shown as diamonds. Kit abbreviations: RZ = RiboZero Gold, LX = Lexogen RiboCop, Q = Qiagen GeneRead rRNA Depletion, NE = NEBNext rRNA Depletion, K=Kapa RiboErase, CR = Clontech Ribogone, CZ = SMARTer Pico total RNA. b ) Reads were mapped to exons in UCSC known gene and scored based on strand of alignment. c ) Fraction of reads mapping to mt rRNA shown as in A. *- RiboZero site 3 used standard RiboZero instead of RiboZero Gold

    Article Snippet: Overall, the Lexogen and Takara/Clontech RiboGone kits generally had the most consistent and even performance on both the ERCC and SIRV spike-in controls across their test sites for intact samples.

    Techniques:

    Northern analyses of a few select forward subtracted SSH cDNA clones . RNA isolated from the leaves of the control and 425 mM NaCl-treated plants and blotted onto Hybond N + membrane was hybridized with the individual radiolabelled ESTs. A RNA blot each for the control and NaCl-treated sample was hybridized with PCR amplified radiolabelled actin fragment. The horizontal bars against the individual genes represent increase (in%) in transcripts of the respective genes in response to NaCl treatment of the plant when compared to control. The values were obtained after normalization of the blot intensities of actin for the control and NaCl treated sample.

    Journal: BMC Plant Biology

    Article Title: Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization

    doi: 10.1186/1471-2229-9-69

    Figure Lengend Snippet: Northern analyses of a few select forward subtracted SSH cDNA clones . RNA isolated from the leaves of the control and 425 mM NaCl-treated plants and blotted onto Hybond N + membrane was hybridized with the individual radiolabelled ESTs. A RNA blot each for the control and NaCl-treated sample was hybridized with PCR amplified radiolabelled actin fragment. The horizontal bars against the individual genes represent increase (in%) in transcripts of the respective genes in response to NaCl treatment of the plant when compared to control. The values were obtained after normalization of the blot intensities of actin for the control and NaCl treated sample.

    Article Snippet: Double stranded cDNA was prepared by reverse transcription of 4 μg of the purified mRNA in 20 μl reaction solution following the steps outlined in the cDNA preparation kit (Super SMART PCR cDNA synthesis kit, Clontech, Palo alto, USA).

    Techniques: Northern Blot, Clone Assay, Isolation, Northern blot, Polymerase Chain Reaction, Amplification

    The susceptible phenotype of selected mutant lines . (A) Three-week-old NB, pish mutant lines, NC8589 andND2043, and ttm mutant lines were inoculated with M. oryzae containing avrPish . The photographs were taken 7 days post inoculation. (B) Expression of the Pish gene. Total RNA was extracted from leaf tissues of NB and each mutant. Samples (1 μg) of poly(A) + RNA were separated by gel electrophoresis, blotted, and hybridized with radiolabeled probes as indicated. The Actin probe was used as a loading control. The closed arrowhead indicates the size of the Pish mRNA. The asterisk indicates a non-specific signal detected by the Pish probe.

    Journal: BMC Plant Biology

    Article Title: Unique features of the rice blast resistance Pish locus revealed by large scale retrotransposon-tagging

    doi: 10.1186/1471-2229-10-175

    Figure Lengend Snippet: The susceptible phenotype of selected mutant lines . (A) Three-week-old NB, pish mutant lines, NC8589 andND2043, and ttm mutant lines were inoculated with M. oryzae containing avrPish . The photographs were taken 7 days post inoculation. (B) Expression of the Pish gene. Total RNA was extracted from leaf tissues of NB and each mutant. Samples (1 μg) of poly(A) + RNA were separated by gel electrophoresis, blotted, and hybridized with radiolabeled probes as indicated. The Actin probe was used as a loading control. The closed arrowhead indicates the size of the Pish mRNA. The asterisk indicates a non-specific signal detected by the Pish probe.

    Article Snippet: RNA Analysis Poly(A)+ mRNA was purified using an Oligotex™-dT30 < Super > mRNA Purification kit (Takara) according to the manufacturers' instructions.

    Techniques: Mutagenesis, Expressing, Nucleic Acid Electrophoresis

    Quantitative real-time RT-PCR analysis of Pish expression . Total RNA was isolated from NB leaves at 0, 1, 2, and 3 days after inoculation with incompatible or compatible races of M. oryzae , or after mock inoculations. The samples were quantified using Actin mRNA as a reference. Error bars indicate standard deviations.

    Journal: BMC Plant Biology

    Article Title: Unique features of the rice blast resistance Pish locus revealed by large scale retrotransposon-tagging

    doi: 10.1186/1471-2229-10-175

    Figure Lengend Snippet: Quantitative real-time RT-PCR analysis of Pish expression . Total RNA was isolated from NB leaves at 0, 1, 2, and 3 days after inoculation with incompatible or compatible races of M. oryzae , or after mock inoculations. The samples were quantified using Actin mRNA as a reference. Error bars indicate standard deviations.

    Article Snippet: RNA Analysis Poly(A)+ mRNA was purified using an Oligotex™-dT30 < Super > mRNA Purification kit (Takara) according to the manufacturers' instructions.

    Techniques: Quantitative RT-PCR, Expressing, Isolation

    (A) AAV vector maps depicting the AAV-P Mecp2 -Cas9 vector and an inducible gRNA vector, AAV-gRNAi-TetR-GFP. (B) The gRNAi plasmid described in (A) , containing either a Tet gRNA or no gRNA (Empty), were co-transfected with pX330 Empty into N2A cells in the presence or absence of Dox. Nighty six hours post transfection, the cells were harvested, the genomic DNA was isolated and PCR, and RFLP analysis was performed to assess if genome editing had occurred at the Tet2 locus. Genome editing was not observed in cells that had received the gRNA Empty plasmid either in the presence or absence of Dox as expected. Cells that were transfected with a plasmid containing the gRNA Tet2 plasmid, did exhibit editing in the presence of Dox, but not in the absence of Dox, indicating that genome editing could be regulated in a Dox dependent manner. Similar results were observed in at least 3 independent samples per group. (C) The H1/TO promoter gRNA expression cassette was compared to gRNA expression cassettes composed of either a full length H1/TO promoter (H1-L/TO), a U6/TO promoter or a non-inducible U6 promoter (U6) for their ability to edit the Tet2 locus. Cells were transfected with the respective vectors in the presence or absence of Dox and genome editing was assessed 96 h post transfection ( n = 4 per groups). (D) Quantification of genome editing revealed that the inducible promoter gRNA expression cassettes were not significantly different from each other (n.s. = p ≤ 0.0778) in their ability to edit the Tet2 locus and they were all slightly but significantly different from the non-inducible U6 promoter construct ( * p ≤ 0.003). Four independent samples were screened with similar results. (E) Partial sequences of the inducible promoters used, highlighting the tetracycline operators (Tet-O) and transcriptional start site. (F) Genomic DNA from the same U6, H1/TO, and UTC samples used for (C,D) were subjected to a mutation detection kit (Clontech), in which the PCR amplified product was denatured, annealed and digested with Resolvase to directly detect edited DNA. (G) Levels of editing were comparable to those seen using the restriction digest PCR/RFLP analysis used for (C,D) . Again, levels of editing were significantly higher in the U6 group, compared to the H1/TO group (# p ≤ 0.005, Two Tailed T -test).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

    doi: 10.3389/fnmol.2016.00070

    Figure Lengend Snippet: (A) AAV vector maps depicting the AAV-P Mecp2 -Cas9 vector and an inducible gRNA vector, AAV-gRNAi-TetR-GFP. (B) The gRNAi plasmid described in (A) , containing either a Tet gRNA or no gRNA (Empty), were co-transfected with pX330 Empty into N2A cells in the presence or absence of Dox. Nighty six hours post transfection, the cells were harvested, the genomic DNA was isolated and PCR, and RFLP analysis was performed to assess if genome editing had occurred at the Tet2 locus. Genome editing was not observed in cells that had received the gRNA Empty plasmid either in the presence or absence of Dox as expected. Cells that were transfected with a plasmid containing the gRNA Tet2 plasmid, did exhibit editing in the presence of Dox, but not in the absence of Dox, indicating that genome editing could be regulated in a Dox dependent manner. Similar results were observed in at least 3 independent samples per group. (C) The H1/TO promoter gRNA expression cassette was compared to gRNA expression cassettes composed of either a full length H1/TO promoter (H1-L/TO), a U6/TO promoter or a non-inducible U6 promoter (U6) for their ability to edit the Tet2 locus. Cells were transfected with the respective vectors in the presence or absence of Dox and genome editing was assessed 96 h post transfection ( n = 4 per groups). (D) Quantification of genome editing revealed that the inducible promoter gRNA expression cassettes were not significantly different from each other (n.s. = p ≤ 0.0778) in their ability to edit the Tet2 locus and they were all slightly but significantly different from the non-inducible U6 promoter construct ( * p ≤ 0.003). Four independent samples were screened with similar results. (E) Partial sequences of the inducible promoters used, highlighting the tetracycline operators (Tet-O) and transcriptional start site. (F) Genomic DNA from the same U6, H1/TO, and UTC samples used for (C,D) were subjected to a mutation detection kit (Clontech), in which the PCR amplified product was denatured, annealed and digested with Resolvase to directly detect edited DNA. (G) Levels of editing were comparable to those seen using the restriction digest PCR/RFLP analysis used for (C,D) . Again, levels of editing were significantly higher in the U6 group, compared to the H1/TO group (# p ≤ 0.005, Two Tailed T -test).

    Article Snippet: Some in vitro samples were screened using the resolvase based mutation detection kit (Guide-it Mutation Detection Kit, Clontech, Cat #631443) following the manufacturer's instructions.

    Techniques: Plasmid Preparation, Transfection, Isolation, Polymerase Chain Reaction, Expressing, Construct, Mutagenesis, Amplification, Two Tailed Test