ecorv hf  (New England Biolabs)


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    EcoRV HF
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    EcoRV HF 20 000 units
    Catalog Number:
    R3195L
    Price:
    249
    Category:
    Restriction Enzymes
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    20 000 units
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    New England Biolabs ecorv hf
    EcoRV HF
    EcoRV HF 20 000 units
    https://www.bioz.com/result/ecorv hf/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ecorv hf - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) 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

    2) Product Images from "SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues"

    Article Title: SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

    Journal: bioRxiv

    doi: 10.1101/2020.11.16.385237

    SeqStain based multiplex immunofluorescence imaging. A . Schematic representing the SeqStain methodology. Immobilized cells and tissue sections on glass are processed in cycles of immuno-staining with fluorescent-DNA labelled antibodies (step 1), imaging (step 2), gentle destaining using a nuclease (step 3), re-imaging (step 4) followed by next round of staining steps (step 5). Post-imaging, the data is analysed by computational stacking and alignment of the images followed by analyses of the spatial relationships between various markers to generate spatial maps of molecules and cells. Schematics were generated using Biorender. B . Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. Scale bar is 100μm. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. Graphs show the mean ± standard deviation. C. Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labelled with AF488 fluorophore) or anti-CD45 (labelled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labelled using DAPI. All images are representative of at least three replicates. Scale bar is 100 μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. D. Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 minute after the addition of DNase I (bottom panel). All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. E. Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labelled using the AF488 fluorophore shown in green and the antibodies labelled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is also presented. Graphs show the mean ± standard deviation.
    Figure Legend Snippet: SeqStain based multiplex immunofluorescence imaging. A . Schematic representing the SeqStain methodology. Immobilized cells and tissue sections on glass are processed in cycles of immuno-staining with fluorescent-DNA labelled antibodies (step 1), imaging (step 2), gentle destaining using a nuclease (step 3), re-imaging (step 4) followed by next round of staining steps (step 5). Post-imaging, the data is analysed by computational stacking and alignment of the images followed by analyses of the spatial relationships between various markers to generate spatial maps of molecules and cells. Schematics were generated using Biorender. B . Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. Scale bar is 100μm. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. Graphs show the mean ± standard deviation. C. Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labelled with AF488 fluorophore) or anti-CD45 (labelled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labelled using DAPI. All images are representative of at least three replicates. Scale bar is 100 μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. D. Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 minute after the addition of DNase I (bottom panel). All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. E. Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labelled using the AF488 fluorophore shown in green and the antibodies labelled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is also presented. Graphs show the mean ± standard deviation.

    Techniques Used: Multiplex Assay, Immunofluorescence, Imaging, Immunostaining, Staining, Generated, Fluorescence, Standard Deviation

    3) Product Images from "Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism"

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

    Journal: bioRxiv

    doi: 10.1101/2020.07.20.211623

    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).
    Figure Legend 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).

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

    Related Articles

    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.

    Plasmid Preparation:

    Article Title: Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells
    Article Snippet: Each spike-in sequence contained a T7 promoter for in vitro transcription. .. Approximately 1.5 µg of each plasmid was digested with 10 U of EcoRV-HF (New England BioLabs) and purified with DNA-binding magnetic beads (SPRI-select, Beckman Coulter). .. Approximately, 1 µg of the digested plasmid was then used for in vitro transcription (MEGAscript T7 Transcription Kit, ThermoFisher Scientific).

    Article Title: Stepwise accumulation of mutations in CesA3 in Phytophthora sojae results in increasing resistance to CAA fungicides, et al. Stepwise accumulation of mutations in CesA3 in Phytophthora sojae results in increasing resistance to CAA fungicides
    Article Snippet: .. The transformation vector for the homology directed repair was prepared by cloning a 1305‐bp fragment of the CesA3 gene from the wild‐type P. sojae isolate P6497 into the Eco RV (NEB, R3195S) site of the pBluescript II KS + vector, using the In‐Fusion® HD Cloning Kit (Clontech). .. The primers PsCesA3.HDT.F1 and PsCesA3.HDT.R1 used for the amplification of the 1305‐bp homologous donor template are listed in Table .

    Purification:

    Article Title: Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells
    Article Snippet: Each spike-in sequence contained a T7 promoter for in vitro transcription. .. Approximately 1.5 µg of each plasmid was digested with 10 U of EcoRV-HF (New England BioLabs) and purified with DNA-binding magnetic beads (SPRI-select, Beckman Coulter). .. Approximately, 1 µg of the digested plasmid was then used for in vitro transcription (MEGAscript T7 Transcription Kit, ThermoFisher Scientific).

    Magnetic Beads:

    Article Title: Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells
    Article Snippet: Each spike-in sequence contained a T7 promoter for in vitro transcription. .. Approximately 1.5 µg of each plasmid was digested with 10 U of EcoRV-HF (New England BioLabs) and purified with DNA-binding magnetic beads (SPRI-select, Beckman Coulter). .. Approximately, 1 µg of the digested plasmid was then used for in vitro transcription (MEGAscript T7 Transcription Kit, ThermoFisher Scientific).

    Blocking Assay:

    Article Title: SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues
    Article Snippet: .. Block-1 solution (PBS containing 1% Bovine Serum Albumin (Sigma-Aldrich, #B4287-1G)), Block-2 solution (PBS containing 200ng/ml salmon sperm DNA (Thermo Fisher, #AM9680) and 3 nanomoles/ml singlestranded DNA and 0.5M NaCl), Wash buffer (PBS containing 0.1% Tween-20 (Sigma-Aldrich, #P1379), DNase I de-staining buffer (PBS containing 1X DNase buffer and 20 units of DNase I (NEB, #M0303) in 500ul), EcoRV de-staining buffer (deionized water containing 1X cut-smart buffer and 200 units of EcoRV (NEB, #R3195S) in 500ul), SmaI de-staining buffer (de-ionized water containing 1X cut smart buffer and 200 units of SmaI (NEB, #R01041S) in 500ul). .. Preparation of cells for SeqStain25 mm round cover glass (Fisher Brand, #12-545-102 25CIR-1) was coated with 3-Triethoxysilypropylamine APES (Millipore, #A3648) following manufacturer’s instructions.

    Clone Assay:

    Article Title: Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism
    Article Snippet: Colony PCR was performed using oAH195-196 to verify the presence of the insert, and positive clones were confirmed by Sanger sequencing. .. 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. .. To prepare viral gene inserts, 1 μg of pUC containing the relevant gene was initially digested with AvrII in the same reaction conditions followed by purification.

    Article Title: Stepwise accumulation of mutations in CesA3 in Phytophthora sojae results in increasing resistance to CAA fungicides, et al. Stepwise accumulation of mutations in CesA3 in Phytophthora sojae results in increasing resistance to CAA fungicides
    Article Snippet: .. The transformation vector for the homology directed repair was prepared by cloning a 1305‐bp fragment of the CesA3 gene from the wild‐type P. sojae isolate P6497 into the Eco RV (NEB, R3195S) site of the pBluescript II KS + vector, using the In‐Fusion® HD Cloning Kit (Clontech). .. The primers PsCesA3.HDT.F1 and PsCesA3.HDT.R1 used for the amplification of the 1305‐bp homologous donor template are listed in Table .

    DNA Purification:

    Article Title: Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism
    Article Snippet: Colony PCR was performed using oAH195-196 to verify the presence of the insert, and positive clones were confirmed by Sanger sequencing. .. 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. .. To prepare viral gene inserts, 1 μg of pUC containing the relevant gene was initially digested with AvrII in the same reaction conditions followed by purification.

    Transformation Assay:

    Article Title: Stepwise accumulation of mutations in CesA3 in Phytophthora sojae results in increasing resistance to CAA fungicides, et al. Stepwise accumulation of mutations in CesA3 in Phytophthora sojae results in increasing resistance to CAA fungicides
    Article Snippet: .. The transformation vector for the homology directed repair was prepared by cloning a 1305‐bp fragment of the CesA3 gene from the wild‐type P. sojae isolate P6497 into the Eco RV (NEB, R3195S) site of the pBluescript II KS + vector, using the In‐Fusion® HD Cloning Kit (Clontech). .. The primers PsCesA3.HDT.F1 and PsCesA3.HDT.R1 used for the amplification of the 1305‐bp homologous donor template are listed in Table .

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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 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/spei ecorv hf/product/New England Biolabs
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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

    SeqStain based multiplex immunofluorescence imaging. A . Schematic representing the SeqStain methodology. Immobilized cells and tissue sections on glass are processed in cycles of immuno-staining with fluorescent-DNA labelled antibodies (step 1), imaging (step 2), gentle destaining using a nuclease (step 3), re-imaging (step 4) followed by next round of staining steps (step 5). Post-imaging, the data is analysed by computational stacking and alignment of the images followed by analyses of the spatial relationships between various markers to generate spatial maps of molecules and cells. Schematics were generated using Biorender. B . Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. Scale bar is 100μm. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. Graphs show the mean ± standard deviation. C. Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labelled with AF488 fluorophore) or anti-CD45 (labelled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labelled using DAPI. All images are representative of at least three replicates. Scale bar is 100 μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. D. Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 minute after the addition of DNase I (bottom panel). All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. E. Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labelled using the AF488 fluorophore shown in green and the antibodies labelled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is also presented. Graphs show the mean ± standard deviation.

    Journal: bioRxiv

    Article Title: SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

    doi: 10.1101/2020.11.16.385237

    Figure Lengend Snippet: SeqStain based multiplex immunofluorescence imaging. A . Schematic representing the SeqStain methodology. Immobilized cells and tissue sections on glass are processed in cycles of immuno-staining with fluorescent-DNA labelled antibodies (step 1), imaging (step 2), gentle destaining using a nuclease (step 3), re-imaging (step 4) followed by next round of staining steps (step 5). Post-imaging, the data is analysed by computational stacking and alignment of the images followed by analyses of the spatial relationships between various markers to generate spatial maps of molecules and cells. Schematics were generated using Biorender. B . Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. Scale bar is 100μm. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. Graphs show the mean ± standard deviation. C. Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labelled with AF488 fluorophore) or anti-CD45 (labelled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labelled using DAPI. All images are representative of at least three replicates. Scale bar is 100 μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. D. Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 minute after the addition of DNase I (bottom panel). All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. E. Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labelled using the AF488 fluorophore shown in green and the antibodies labelled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is also presented. Graphs show the mean ± standard deviation.

    Article Snippet: Block-1 solution (PBS containing 1% Bovine Serum Albumin (Sigma-Aldrich, #B4287-1G)), Block-2 solution (PBS containing 200ng/ml salmon sperm DNA (Thermo Fisher, #AM9680) and 3 nanomoles/ml singlestranded DNA and 0.5M NaCl), Wash buffer (PBS containing 0.1% Tween-20 (Sigma-Aldrich, #P1379), DNase I de-staining buffer (PBS containing 1X DNase buffer and 20 units of DNase I (NEB, #M0303) in 500ul), EcoRV de-staining buffer (deionized water containing 1X cut-smart buffer and 200 units of EcoRV (NEB, #R3195S) in 500ul), SmaI de-staining buffer (de-ionized water containing 1X cut smart buffer and 200 units of SmaI (NEB, #R01041S) in 500ul).

    Techniques: Multiplex Assay, Immunofluorescence, Imaging, Immunostaining, Staining, Generated, Fluorescence, Standard Deviation

    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