spei ecorv hf  (New England Biolabs)


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    EcoRV HF
    Description:
    EcoRV HF 20 000 units
    Catalog Number:
    r3195l
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    249
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    20 000 units
    Category:
    Restriction Enzymes
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    New England Biolabs spei ecorv hf
    EcoRV HF
    EcoRV HF 20 000 units
    https://www.bioz.com/result/spei ecorv hf/product/New England Biolabs
    Average 99 stars, based on 79 article reviews
    Price from $9.99 to $1999.99
    spei ecorv hf - by Bioz Stars, 2020-08
    99/100 stars

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    1) Product Images from "A single epidermal stem cell strategy for safe ex vivo gene therapy"

    Article Title: A single epidermal stem cell strategy for safe ex vivo gene therapy

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201404353

    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
    Figure Legend Snippet: Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.

    Techniques Used: Isolation, Infection, Clone Assay, Immunostaining, Expressing, Staining, Irradiation, Quantitative RT-PCR, Southern Blot, Western Blot, Negative Control, Positive Control

    2) Product Images from "The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing"

    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

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2016.00070

    (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.
    Figure Legend Snippet: (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    Techniques Used: Plasmid Preparation, Expressing, Immunocytochemistry, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Transfection, Isolation, Purification, Clone Assay

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    Amplification:

    Article Title: TRPV6 Variants Interfere with Maternal-Fetal Calcium Transport through the Placenta and Cause Transient Neonatal Hyperparathyroidism
    Article Snippet: .. Amplified fragments were treated with T4 polynucleotide kinase (Takara, Japan), ligated by T4 ligase (ToYoBo, Japan) with pcDNA3.1 (+) (Life Technologies, USA), and pretreated with Eco R V-HF (NEB, USA) and CIP (Takara, Japan). ..

    Ligation:

    Article Title: Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe
    Article Snippet: .. Identification of insertion sites by inverse splinkerette PCR Genomic DNA (10 μg) was digested with 80 units of EcoR V-HF (NEB) at 37°C for 16 h, followed by 65°C denaturation for 15 min. A total of 1.5 μg of digested DNA was used in a 500-μl ligation (final DNA concentration = 3 ng/μl) with 2000 units of T4 DNA ligase at 16°C for 16 h. Ligated DNA was subsequently digested with 60 units of Sfi I restriction endonuclease at 50°C for five hours. .. All of the Sfi I-digested DNA was used in a 30-μl ligation reaction that contained 0.33 μM of double-strand Sfi I splinkerette and 800 units of T4 DNA ligase.

    Ethanol Precipitation:

    Article Title: Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions
    Article Snippet: .. After phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation, the recovered plasmids were treated with or without Eco RV-HF (New England Biolabs). .. E. coli strain NEB10β (New England Biolabs) was transformed with the mixture of above plasmids and 0.08 ng of pZeo, which serves as an internal control for transformation efficiency, and then plated onto 1xYT plates with carbenicillin/blasticidin S and 1xYT plates with zeocin, respectively.

    Concentration Assay:

    Article Title: Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe
    Article Snippet: .. Identification of insertion sites by inverse splinkerette PCR Genomic DNA (10 μg) was digested with 80 units of EcoR V-HF (NEB) at 37°C for 16 h, followed by 65°C denaturation for 15 min. A total of 1.5 μg of digested DNA was used in a 500-μl ligation (final DNA concentration = 3 ng/μl) with 2000 units of T4 DNA ligase at 16°C for 16 h. Ligated DNA was subsequently digested with 60 units of Sfi I restriction endonuclease at 50°C for five hours. .. All of the Sfi I-digested DNA was used in a 30-μl ligation reaction that contained 0.33 μM of double-strand Sfi I splinkerette and 800 units of T4 DNA ligase.

    Luciferase:

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ). .. Nucleofection Early passage MSCs (1 × 106 cells) were nucleofected with 5 μ g pVITRO2-Luc2 and 2 μ g pmax-GFP® , according to the manufacturer's instructions (Lonza™, Basel, Switzerland), using the Nucleofector™ 2b device (Lonza™).

    Expressing:

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ). .. Nucleofection Early passage MSCs (1 × 106 cells) were nucleofected with 5 μ g pVITRO2-Luc2 and 2 μ g pmax-GFP® , according to the manufacturer's instructions (Lonza™, Basel, Switzerland), using the Nucleofector™ 2b device (Lonza™).

    Polymerase Chain Reaction:

    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
    Article Snippet: .. Three microliters of PCR product was digested with 5 units of EcoRV-HF (New England Biolabs) for 2 h in a standard 10 μl restriction enzyme reaction following the manufacturer's instructions. .. Each 3 μL of digested PCR product was electrophoresed on a 2.0% agarose, 0.5 X TBE gel to determine if genome editing had occurred.

    Article Title: Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe
    Article Snippet: .. Identification of insertion sites by inverse splinkerette PCR Genomic DNA (10 μg) was digested with 80 units of EcoR V-HF (NEB) at 37°C for 16 h, followed by 65°C denaturation for 15 min. A total of 1.5 μg of digested DNA was used in a 500-μl ligation (final DNA concentration = 3 ng/μl) with 2000 units of T4 DNA ligase at 16°C for 16 h. Ligated DNA was subsequently digested with 60 units of Sfi I restriction endonuclease at 50°C for five hours. .. All of the Sfi I-digested DNA was used in a 30-μl ligation reaction that contained 0.33 μM of double-strand Sfi I splinkerette and 800 units of T4 DNA ligase.

    Plasmid Preparation:

    Article Title: A transcriptional circuit filters oscillating circadian hormonal inputs to regulate fat cell differentiation
    Article Snippet: .. The pENTR1a backbone vector was digested with EcoR Ι-HF and BamH Ι-HF (NEB), and assembled together with three DNA fragments coding for homology arm 1, Citrine or mKate2, and homology arm 2 using Gibson assembly. .. The homology arm fragments were PCR amplified from OP9 genomic DNA with primers introducing a 15–20 bp overhang used for Gibson assembly.

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ). .. Nucleofection Early passage MSCs (1 × 106 cells) were nucleofected with 5 μ g pVITRO2-Luc2 and 2 μ g pmax-GFP® , according to the manufacturer's instructions (Lonza™, Basel, Switzerland), using the Nucleofector™ 2b device (Lonza™).

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    New England Biolabs spei ecorv hf
    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with <t>EcoRV</t> and <t>SpeI</t> that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
    Spei Ecorv Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/spei ecorv hf/product/New England Biolabs
    Average 99 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    spei ecorv hf - by Bioz Stars, 2020-08
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    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.

    Journal: EMBO Molecular Medicine

    Article Title: A single epidermal stem cell strategy for safe ex vivo gene therapy

    doi: 10.15252/emmm.201404353

    Figure Lengend Snippet: Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.

    Article Snippet: Ten micrograms of DNA was codigested with SpeI/EcoRV HF (New England Biolabs) and loaded on a 0.8% agarose (Promega) gel.

    Techniques: Isolation, Infection, Clone Assay, Immunostaining, Expressing, Staining, Irradiation, Quantitative RT-PCR, Southern Blot, Western Blot, Negative Control, Positive Control

    (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    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 AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    Article Snippet: Three microliters of PCR product was digested with 5 units of EcoRV-HF (New England Biolabs) for 2 h in a standard 10 μl restriction enzyme reaction following the manufacturer's instructions.

    Techniques: Plasmid Preparation, Expressing, Immunocytochemistry, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Transfection, Isolation, Purification, Clone Assay