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  • 99
    New England Biolabs ecorv hf endonuclease
    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.
    Ecorv Hf Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 29 article reviews
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    ecorv hf endonuclease - by Bioz Stars, 2020-08
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    89
    New England Biolabs ecorv hf
    Schematics illustrating the cloning strategy. (A) The three parental vectors used for generating untagged, N-, and C-terminally tagged constructs. (B) Features common to all synthesized insert sequences. Each insert included <t>BamHI</t> and <t>EcoRV</t> sites at either end to facilitate cloning into the three parental vectors. To allow for C-terminal cloning, an AvrII site was inserted such that it overlapped the stop codon (see text for details). In order to accommodate the AvrII site, an alanine residue was added to the end of each expression construct. The viral ORFs were codon-optimized for moderate or high expression, and lacked BamHI, AvrII, and EcoRV sites. (C) For untagged and N-terminal tagging, inserts were digested with BamHI and EcoRV and ligated directly into plasmid precut with the same enzymes. For C-terminal cloning, the inserts were first digested with AvrII, blunted with Mung Bean Nuclease, and then cut with BamHI. The resulting fragment was ligated into plasmid cut with BamHI and EcoRV. (D) Untagged, N-, and C-terminally tagged expression constructs. (E) Strategy for generating stable cell lines (see text for details).
    Ecorv Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecorv hf/product/New England Biolabs
    Average 89 stars, based on 195 article reviews
    Price from $9.99 to $1999.99
    ecorv hf - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

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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

    Schematics illustrating the cloning strategy. (A) The three parental vectors used for generating untagged, N-, and C-terminally tagged constructs. (B) Features common to all synthesized insert sequences. Each insert included BamHI and EcoRV sites at either end to facilitate cloning into the three parental vectors. To allow for C-terminal cloning, an AvrII site was inserted such that it overlapped the stop codon (see text for details). In order to accommodate the AvrII site, an alanine residue was added to the end of each expression construct. The viral ORFs were codon-optimized for moderate or high expression, and lacked BamHI, AvrII, and EcoRV sites. (C) For untagged and N-terminal tagging, inserts were digested with BamHI and EcoRV and ligated directly into plasmid precut with the same enzymes. For C-terminal cloning, the inserts were first digested with AvrII, blunted with Mung Bean Nuclease, and then cut with BamHI. The resulting fragment was ligated into plasmid cut with BamHI and EcoRV. (D) Untagged, N-, and C-terminally tagged expression constructs. (E) Strategy for generating stable cell lines (see text for details).

    Journal: bioRxiv

    Article Title: Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism

    doi: 10.1101/2020.07.20.211623

    Figure Lengend Snippet: Schematics illustrating the cloning strategy. (A) The three parental vectors used for generating untagged, N-, and C-terminally tagged constructs. (B) Features common to all synthesized insert sequences. Each insert included BamHI and EcoRV sites at either end to facilitate cloning into the three parental vectors. To allow for C-terminal cloning, an AvrII site was inserted such that it overlapped the stop codon (see text for details). In order to accommodate the AvrII site, an alanine residue was added to the end of each expression construct. The viral ORFs were codon-optimized for moderate or high expression, and lacked BamHI, AvrII, and EcoRV sites. (C) For untagged and N-terminal tagging, inserts were digested with BamHI and EcoRV and ligated directly into plasmid precut with the same enzymes. For C-terminal cloning, the inserts were first digested with AvrII, blunted with Mung Bean Nuclease, and then cut with BamHI. The resulting fragment was ligated into plasmid cut with BamHI and EcoRV. (D) Untagged, N-, and C-terminally tagged expression constructs. (E) Strategy for generating stable cell lines (see text for details).

    Article Snippet: Cloning of C-terminally tagged viral genesTo prepare the backbone, 2 μg of pcDNA5-FRT-TO-C-His8-Flag was digested with BamHI-HF and EcoRV-HF, as above, followed by DNA purification.

    Techniques: Clone Assay, Construct, Synthesized, Expressing, Plasmid Preparation, Stable Transfection