tie2 expression  (Sino Biological)


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    Name:
    TIE2 cDNA ORF Clone Mouse untagged
    Description:
    Full length Clone DNA of Mouse endothelial specific receptor tyrosine kinase
    Catalog Number:
    MG51087-UT
    Price:
    295.0
    Category:
    cDNA Clone
    Applications:
    Stable or Transient mammalian expression
    Size:
    1Unit
    Product Aliases:
    AA517024 cDNA ORF Clone Mouse, Cd202b cDNA ORF Clone Mouse, Hyk cDNA ORF Clone Mouse, STK1 cDNA ORF Clone Mouse, Tie-2 cDNA ORF Clone Mouse, Tie2 cDNA ORF Clone Mouse
    Molecule Name:
    TEK,Tie2,
    Buy from Supplier


    Structured Review

    Sino Biological tie2 expression
    TIE2 cDNA ORF Clone Mouse untagged
    Full length Clone DNA of Mouse endothelial specific receptor tyrosine kinase
    https://www.bioz.com/result/tie2 expression/product/Sino Biological
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tie2 expression - by Bioz Stars, 2021-07
    90/100 stars

    Images

    1) Product Images from "Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells"

    Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

    Journal: Scientific Reports

    doi: 10.1038/srep42127

    Analysis of CRISPR-Cas9 induced Tie2 gene mutations using next-generation sequencing. ( A ) Indel size distribution of wild-type cells (control) and cells transduced with two different sgRNAs targeting Tie2 (sgRNA Tie2-1, sgRNA Tie2-2). ( B ) NHEJ reads with insertions, deletions, and substitutions were mapped to reference amplicon position. Sequencing/alignment errors (green lines) can be distinguished from indels by their similar positions in all three samples. ( C ) Frameshift mutagenesis profile and predicted Cas9 cleavage site. Unmodified reads are excluded from this analysis. sgRNA Tie2-1 and sgRNA Tie2-2 showed different percentage of reads with mutations from frameshift, in frame, and in noncoding region. ( D ) Predicted impacts on splice sites. Potential splice sites modified refers to the reads in which either of the two introns adjacent to the exon is disrupted. ( E ) The relative contributions of potentially disruptive coding region mutations (indels) and non-coding region mutations (5′ splice site) were quantified across 3 passages. SNPs, in-frame indels, indels less than 5 residues, and 3′ splice site mutations were excluded.
    Figure Legend Snippet: Analysis of CRISPR-Cas9 induced Tie2 gene mutations using next-generation sequencing. ( A ) Indel size distribution of wild-type cells (control) and cells transduced with two different sgRNAs targeting Tie2 (sgRNA Tie2-1, sgRNA Tie2-2). ( B ) NHEJ reads with insertions, deletions, and substitutions were mapped to reference amplicon position. Sequencing/alignment errors (green lines) can be distinguished from indels by their similar positions in all three samples. ( C ) Frameshift mutagenesis profile and predicted Cas9 cleavage site. Unmodified reads are excluded from this analysis. sgRNA Tie2-1 and sgRNA Tie2-2 showed different percentage of reads with mutations from frameshift, in frame, and in noncoding region. ( D ) Predicted impacts on splice sites. Potential splice sites modified refers to the reads in which either of the two introns adjacent to the exon is disrupted. ( E ) The relative contributions of potentially disruptive coding region mutations (indels) and non-coding region mutations (5′ splice site) were quantified across 3 passages. SNPs, in-frame indels, indels less than 5 residues, and 3′ splice site mutations were excluded.

    Techniques Used: CRISPR, Next-Generation Sequencing, Transduction, Non-Homologous End Joining, Amplification, Sequencing, Mutagenesis, Modification

    Deletion of Tie2 gene in primary ECs using CRISPR-Cas9. ( A ) Cultured primary HLMVECs were transduced by EGFP-Cas9 adenovirus and sgRNA lentivirus targeting on Tie2 and subjected to flow cytometry analysis of EGFP expression. In a separate study, HLMVECs were transduced by GFP lentivirus and subjected to flow cytometry analysis of EGFP expression as an indicator of lentivirus transduction efficiency. ( B ) Protein expression of Tie2 in vector control and Tie2 knockout HLMVECs induced by CRISPR-Cas9 was determined by immunoblotting. Overexpression of mouse Tie2 in Tie2 −/− HLMVECs was determined to assess the ability to rescue Tie2 expression after deletion. The uncropped full-length gels can be found in Supplementary Fig. S1 . ( C ) Quantification of Tie2 protein expression from 3 independent experiments. sgRNA Tie2-1 and sgRNA Tie2-2 are CRISPR-Cas9-mediated deletions of Tie-2 at two distinction domains of Tie2 (two different sgRNAs). HuTie2KO1 and HuTie2KO2 + mTie2 represent restoration of Tie-2 expression in cells having undergone Tie-2 deletion. Differences were calculated using one-way ANOVA. P values less than 0.05 are indicated in the graph. ( D ) T7E1 assay detecting mutation on HLMVECs edited by CRISPR-Cas9 with Tie2 sgRNAs as showing a series of bands. Wild-types cells with or without Cas9 or vector only show single bands (negative controls).
    Figure Legend Snippet: Deletion of Tie2 gene in primary ECs using CRISPR-Cas9. ( A ) Cultured primary HLMVECs were transduced by EGFP-Cas9 adenovirus and sgRNA lentivirus targeting on Tie2 and subjected to flow cytometry analysis of EGFP expression. In a separate study, HLMVECs were transduced by GFP lentivirus and subjected to flow cytometry analysis of EGFP expression as an indicator of lentivirus transduction efficiency. ( B ) Protein expression of Tie2 in vector control and Tie2 knockout HLMVECs induced by CRISPR-Cas9 was determined by immunoblotting. Overexpression of mouse Tie2 in Tie2 −/− HLMVECs was determined to assess the ability to rescue Tie2 expression after deletion. The uncropped full-length gels can be found in Supplementary Fig. S1 . ( C ) Quantification of Tie2 protein expression from 3 independent experiments. sgRNA Tie2-1 and sgRNA Tie2-2 are CRISPR-Cas9-mediated deletions of Tie-2 at two distinction domains of Tie2 (two different sgRNAs). HuTie2KO1 and HuTie2KO2 + mTie2 represent restoration of Tie-2 expression in cells having undergone Tie-2 deletion. Differences were calculated using one-way ANOVA. P values less than 0.05 are indicated in the graph. ( D ) T7E1 assay detecting mutation on HLMVECs edited by CRISPR-Cas9 with Tie2 sgRNAs as showing a series of bands. Wild-types cells with or without Cas9 or vector only show single bands (negative controls).

    Techniques Used: CRISPR, Cell Culture, Flow Cytometry, Expressing, Transduction, Plasmid Preparation, Knock-Out, Over Expression, Mutagenesis

    Tie2 deletion by CRISPR-Cas9 in primary ECs increases endothelial permeability and mitigates recovery of permeability in response to thrombin challenge. ( A ) Basal TER and post-thrombin (1 U/ml) TER were studied in confluent control, Tie2-deleted HLMVECs and mTie2 overexpressing cells in which Tie2 had been deleted. Absolute TER values were reduced in both Tie2-deleted groups as compared to control ECs at basal condition. mTie2 overexpression successfully rescued the basal leakiness. ( B ) Quantification of TER values of wild-type (control), transduced cells (sgRNA Tie2-1, sgRNA Tie2-2) and rescued cells (HuTie2KO + mTie2) at basal (−1 h), thrombin-stimulated (0.5 h) and post-recovery (3 h) condition. Differences were calculated using two-way ANOVA. P values less than 0.05 are indicated in the graph. n = 3. ( C ) Serum-starved confluent control or Tie2-deleted HLMVECs were challenged by PBS or 1 U/ml of thrombin, and subjected for VE-cadherin immunostaining at the indicated time-points and analyzed by confocal microscopy. The marked disruption of VE-cadherin junctions seen in wild-type HLMVEC monolayer (control) at the 30 min post thrombin (white arrows) was reversed by 2 h; however, the defective VE-cadherin junctions were present in Tie2-deleted HLMVECs 2 h post-thrombin. White arrows are used to identify areas of adherens junction disruption where neighboring cells lack cell membrane localization of VE-cadherin. Results are representative of 3 independent experiments.
    Figure Legend Snippet: Tie2 deletion by CRISPR-Cas9 in primary ECs increases endothelial permeability and mitigates recovery of permeability in response to thrombin challenge. ( A ) Basal TER and post-thrombin (1 U/ml) TER were studied in confluent control, Tie2-deleted HLMVECs and mTie2 overexpressing cells in which Tie2 had been deleted. Absolute TER values were reduced in both Tie2-deleted groups as compared to control ECs at basal condition. mTie2 overexpression successfully rescued the basal leakiness. ( B ) Quantification of TER values of wild-type (control), transduced cells (sgRNA Tie2-1, sgRNA Tie2-2) and rescued cells (HuTie2KO + mTie2) at basal (−1 h), thrombin-stimulated (0.5 h) and post-recovery (3 h) condition. Differences were calculated using two-way ANOVA. P values less than 0.05 are indicated in the graph. n = 3. ( C ) Serum-starved confluent control or Tie2-deleted HLMVECs were challenged by PBS or 1 U/ml of thrombin, and subjected for VE-cadherin immunostaining at the indicated time-points and analyzed by confocal microscopy. The marked disruption of VE-cadherin junctions seen in wild-type HLMVEC monolayer (control) at the 30 min post thrombin (white arrows) was reversed by 2 h; however, the defective VE-cadherin junctions were present in Tie2-deleted HLMVECs 2 h post-thrombin. White arrows are used to identify areas of adherens junction disruption where neighboring cells lack cell membrane localization of VE-cadherin. Results are representative of 3 independent experiments.

    Techniques Used: CRISPR, Permeability, Over Expression, Immunostaining, Confocal Microscopy

    Related Articles

    Expressing:

    Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells
    Article Snippet: Western blot pictures were taken using ImageQuant LAS 4000 (GE Healthcare). .. Rescue of Tie2 expression in HLMVECs following Tie-2 deletion Mouse Tie2 (mTie2) cDNA plasmids was purchased from Sino Biological Inc. (# MG51087-G). .. To produce the overexpression adenovirus, the mTie2 cDNA was sub-cloned into pShuttle-CMV vector (a gift from Bert Vogelstein (Addgene plasmid # 16403)) and subsequently recombined into pAdEasy-1 vector (Agilent Technologies) by co-transforming with the BJ5183 cells.

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    Sino Biological tie2 expression
    Analysis of CRISPR-Cas9 induced <t>Tie2</t> gene mutations using next-generation sequencing. ( A ) Indel size distribution of wild-type cells (control) and cells transduced with two different sgRNAs targeting Tie2 (sgRNA Tie2-1, sgRNA Tie2-2). ( B ) NHEJ reads with insertions, deletions, and substitutions were mapped to reference amplicon position. Sequencing/alignment errors (green lines) can be distinguished from indels by their similar positions in all three samples. ( C ) Frameshift mutagenesis profile and predicted Cas9 cleavage site. Unmodified reads are excluded from this analysis. sgRNA Tie2-1 and sgRNA Tie2-2 showed different percentage of reads with mutations from frameshift, in frame, and in noncoding region. ( D ) Predicted impacts on splice sites. Potential splice sites modified refers to the reads in which either of the two introns adjacent to the exon is disrupted. ( E ) The relative contributions of potentially disruptive coding region mutations (indels) and non-coding region mutations (5′ splice site) were quantified across 3 passages. SNPs, in-frame indels, indels less than 5 residues, and 3′ splice site mutations were excluded.
    Tie2 Expression, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tie2 expression/product/Sino Biological
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tie2 expression - by Bioz Stars, 2021-07
    90/100 stars
      Buy from Supplier

    90
    Sino Biological tie 2 deletion mouse tie2 mtie2 cdna plasmids
    Deletion of <t>Tie2</t> gene in primary ECs using CRISPR-Cas9. ( A ) Cultured primary HLMVECs were transduced by EGFP-Cas9 adenovirus and sgRNA lentivirus targeting on Tie2 and subjected to flow cytometry analysis of EGFP expression. In a separate study, HLMVECs were transduced by GFP lentivirus and subjected to flow cytometry analysis of EGFP expression as an indicator of lentivirus transduction efficiency. ( B ) Protein expression of Tie2 in vector control and Tie2 knockout HLMVECs induced by CRISPR-Cas9 was determined by immunoblotting. Overexpression of mouse Tie2 in Tie2 −/− HLMVECs was determined to assess the ability to rescue Tie2 expression after deletion. The uncropped full-length gels can be found in Supplementary Fig. S1 . ( C ) Quantification of Tie2 protein expression from 3 independent experiments. sgRNA Tie2-1 and sgRNA Tie2-2 are CRISPR-Cas9-mediated deletions of <t>Tie-2</t> at two distinction domains of Tie2 (two different sgRNAs). HuTie2KO1 and HuTie2KO2 + mTie2 represent restoration of Tie-2 expression in cells having undergone Tie-2 deletion. Differences were calculated using one-way ANOVA. P values less than 0.05 are indicated in the graph. ( D ) T7E1 assay detecting mutation on HLMVECs edited by CRISPR-Cas9 with Tie2 sgRNAs as showing a series of bands. Wild-types cells with or without Cas9 or vector only show single bands (negative controls).
    Tie 2 Deletion Mouse Tie2 Mtie2 Cdna Plasmids, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tie 2 deletion mouse tie2 mtie2 cdna plasmids/product/Sino Biological
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tie 2 deletion mouse tie2 mtie2 cdna plasmids - by Bioz Stars, 2021-07
    90/100 stars
      Buy from Supplier

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    Analysis of CRISPR-Cas9 induced Tie2 gene mutations using next-generation sequencing. ( A ) Indel size distribution of wild-type cells (control) and cells transduced with two different sgRNAs targeting Tie2 (sgRNA Tie2-1, sgRNA Tie2-2). ( B ) NHEJ reads with insertions, deletions, and substitutions were mapped to reference amplicon position. Sequencing/alignment errors (green lines) can be distinguished from indels by their similar positions in all three samples. ( C ) Frameshift mutagenesis profile and predicted Cas9 cleavage site. Unmodified reads are excluded from this analysis. sgRNA Tie2-1 and sgRNA Tie2-2 showed different percentage of reads with mutations from frameshift, in frame, and in noncoding region. ( D ) Predicted impacts on splice sites. Potential splice sites modified refers to the reads in which either of the two introns adjacent to the exon is disrupted. ( E ) The relative contributions of potentially disruptive coding region mutations (indels) and non-coding region mutations (5′ splice site) were quantified across 3 passages. SNPs, in-frame indels, indels less than 5 residues, and 3′ splice site mutations were excluded.

    Journal: Scientific Reports

    Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

    doi: 10.1038/srep42127

    Figure Lengend Snippet: Analysis of CRISPR-Cas9 induced Tie2 gene mutations using next-generation sequencing. ( A ) Indel size distribution of wild-type cells (control) and cells transduced with two different sgRNAs targeting Tie2 (sgRNA Tie2-1, sgRNA Tie2-2). ( B ) NHEJ reads with insertions, deletions, and substitutions were mapped to reference amplicon position. Sequencing/alignment errors (green lines) can be distinguished from indels by their similar positions in all three samples. ( C ) Frameshift mutagenesis profile and predicted Cas9 cleavage site. Unmodified reads are excluded from this analysis. sgRNA Tie2-1 and sgRNA Tie2-2 showed different percentage of reads with mutations from frameshift, in frame, and in noncoding region. ( D ) Predicted impacts on splice sites. Potential splice sites modified refers to the reads in which either of the two introns adjacent to the exon is disrupted. ( E ) The relative contributions of potentially disruptive coding region mutations (indels) and non-coding region mutations (5′ splice site) were quantified across 3 passages. SNPs, in-frame indels, indels less than 5 residues, and 3′ splice site mutations were excluded.

    Article Snippet: Rescue of Tie2 expression in HLMVECs following Tie-2 deletion Mouse Tie2 (mTie2) cDNA plasmids was purchased from Sino Biological Inc. (# MG51087-G).

    Techniques: CRISPR, Next-Generation Sequencing, Transduction, Non-Homologous End Joining, Amplification, Sequencing, Mutagenesis, Modification

    Deletion of Tie2 gene in primary ECs using CRISPR-Cas9. ( A ) Cultured primary HLMVECs were transduced by EGFP-Cas9 adenovirus and sgRNA lentivirus targeting on Tie2 and subjected to flow cytometry analysis of EGFP expression. In a separate study, HLMVECs were transduced by GFP lentivirus and subjected to flow cytometry analysis of EGFP expression as an indicator of lentivirus transduction efficiency. ( B ) Protein expression of Tie2 in vector control and Tie2 knockout HLMVECs induced by CRISPR-Cas9 was determined by immunoblotting. Overexpression of mouse Tie2 in Tie2 −/− HLMVECs was determined to assess the ability to rescue Tie2 expression after deletion. The uncropped full-length gels can be found in Supplementary Fig. S1 . ( C ) Quantification of Tie2 protein expression from 3 independent experiments. sgRNA Tie2-1 and sgRNA Tie2-2 are CRISPR-Cas9-mediated deletions of Tie-2 at two distinction domains of Tie2 (two different sgRNAs). HuTie2KO1 and HuTie2KO2 + mTie2 represent restoration of Tie-2 expression in cells having undergone Tie-2 deletion. Differences were calculated using one-way ANOVA. P values less than 0.05 are indicated in the graph. ( D ) T7E1 assay detecting mutation on HLMVECs edited by CRISPR-Cas9 with Tie2 sgRNAs as showing a series of bands. Wild-types cells with or without Cas9 or vector only show single bands (negative controls).

    Journal: Scientific Reports

    Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

    doi: 10.1038/srep42127

    Figure Lengend Snippet: Deletion of Tie2 gene in primary ECs using CRISPR-Cas9. ( A ) Cultured primary HLMVECs were transduced by EGFP-Cas9 adenovirus and sgRNA lentivirus targeting on Tie2 and subjected to flow cytometry analysis of EGFP expression. In a separate study, HLMVECs were transduced by GFP lentivirus and subjected to flow cytometry analysis of EGFP expression as an indicator of lentivirus transduction efficiency. ( B ) Protein expression of Tie2 in vector control and Tie2 knockout HLMVECs induced by CRISPR-Cas9 was determined by immunoblotting. Overexpression of mouse Tie2 in Tie2 −/− HLMVECs was determined to assess the ability to rescue Tie2 expression after deletion. The uncropped full-length gels can be found in Supplementary Fig. S1 . ( C ) Quantification of Tie2 protein expression from 3 independent experiments. sgRNA Tie2-1 and sgRNA Tie2-2 are CRISPR-Cas9-mediated deletions of Tie-2 at two distinction domains of Tie2 (two different sgRNAs). HuTie2KO1 and HuTie2KO2 + mTie2 represent restoration of Tie-2 expression in cells having undergone Tie-2 deletion. Differences were calculated using one-way ANOVA. P values less than 0.05 are indicated in the graph. ( D ) T7E1 assay detecting mutation on HLMVECs edited by CRISPR-Cas9 with Tie2 sgRNAs as showing a series of bands. Wild-types cells with or without Cas9 or vector only show single bands (negative controls).

    Article Snippet: Rescue of Tie2 expression in HLMVECs following Tie-2 deletion Mouse Tie2 (mTie2) cDNA plasmids was purchased from Sino Biological Inc. (# MG51087-G).

    Techniques: CRISPR, Cell Culture, Flow Cytometry, Expressing, Transduction, Plasmid Preparation, Knock-Out, Over Expression, Mutagenesis

    Tie2 deletion by CRISPR-Cas9 in primary ECs increases endothelial permeability and mitigates recovery of permeability in response to thrombin challenge. ( A ) Basal TER and post-thrombin (1 U/ml) TER were studied in confluent control, Tie2-deleted HLMVECs and mTie2 overexpressing cells in which Tie2 had been deleted. Absolute TER values were reduced in both Tie2-deleted groups as compared to control ECs at basal condition. mTie2 overexpression successfully rescued the basal leakiness. ( B ) Quantification of TER values of wild-type (control), transduced cells (sgRNA Tie2-1, sgRNA Tie2-2) and rescued cells (HuTie2KO + mTie2) at basal (−1 h), thrombin-stimulated (0.5 h) and post-recovery (3 h) condition. Differences were calculated using two-way ANOVA. P values less than 0.05 are indicated in the graph. n = 3. ( C ) Serum-starved confluent control or Tie2-deleted HLMVECs were challenged by PBS or 1 U/ml of thrombin, and subjected for VE-cadherin immunostaining at the indicated time-points and analyzed by confocal microscopy. The marked disruption of VE-cadherin junctions seen in wild-type HLMVEC monolayer (control) at the 30 min post thrombin (white arrows) was reversed by 2 h; however, the defective VE-cadherin junctions were present in Tie2-deleted HLMVECs 2 h post-thrombin. White arrows are used to identify areas of adherens junction disruption where neighboring cells lack cell membrane localization of VE-cadherin. Results are representative of 3 independent experiments.

    Journal: Scientific Reports

    Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

    doi: 10.1038/srep42127

    Figure Lengend Snippet: Tie2 deletion by CRISPR-Cas9 in primary ECs increases endothelial permeability and mitigates recovery of permeability in response to thrombin challenge. ( A ) Basal TER and post-thrombin (1 U/ml) TER were studied in confluent control, Tie2-deleted HLMVECs and mTie2 overexpressing cells in which Tie2 had been deleted. Absolute TER values were reduced in both Tie2-deleted groups as compared to control ECs at basal condition. mTie2 overexpression successfully rescued the basal leakiness. ( B ) Quantification of TER values of wild-type (control), transduced cells (sgRNA Tie2-1, sgRNA Tie2-2) and rescued cells (HuTie2KO + mTie2) at basal (−1 h), thrombin-stimulated (0.5 h) and post-recovery (3 h) condition. Differences were calculated using two-way ANOVA. P values less than 0.05 are indicated in the graph. n = 3. ( C ) Serum-starved confluent control or Tie2-deleted HLMVECs were challenged by PBS or 1 U/ml of thrombin, and subjected for VE-cadherin immunostaining at the indicated time-points and analyzed by confocal microscopy. The marked disruption of VE-cadherin junctions seen in wild-type HLMVEC monolayer (control) at the 30 min post thrombin (white arrows) was reversed by 2 h; however, the defective VE-cadherin junctions were present in Tie2-deleted HLMVECs 2 h post-thrombin. White arrows are used to identify areas of adherens junction disruption where neighboring cells lack cell membrane localization of VE-cadherin. Results are representative of 3 independent experiments.

    Article Snippet: Rescue of Tie2 expression in HLMVECs following Tie-2 deletion Mouse Tie2 (mTie2) cDNA plasmids was purchased from Sino Biological Inc. (# MG51087-G).

    Techniques: CRISPR, Permeability, Over Expression, Immunostaining, Confocal Microscopy

    Deletion of Tie2 gene in primary ECs using CRISPR-Cas9. ( A ) Cultured primary HLMVECs were transduced by EGFP-Cas9 adenovirus and sgRNA lentivirus targeting on Tie2 and subjected to flow cytometry analysis of EGFP expression. In a separate study, HLMVECs were transduced by GFP lentivirus and subjected to flow cytometry analysis of EGFP expression as an indicator of lentivirus transduction efficiency. ( B ) Protein expression of Tie2 in vector control and Tie2 knockout HLMVECs induced by CRISPR-Cas9 was determined by immunoblotting. Overexpression of mouse Tie2 in Tie2 −/− HLMVECs was determined to assess the ability to rescue Tie2 expression after deletion. The uncropped full-length gels can be found in Supplementary Fig. S1 . ( C ) Quantification of Tie2 protein expression from 3 independent experiments. sgRNA Tie2-1 and sgRNA Tie2-2 are CRISPR-Cas9-mediated deletions of Tie-2 at two distinction domains of Tie2 (two different sgRNAs). HuTie2KO1 and HuTie2KO2 + mTie2 represent restoration of Tie-2 expression in cells having undergone Tie-2 deletion. Differences were calculated using one-way ANOVA. P values less than 0.05 are indicated in the graph. ( D ) T7E1 assay detecting mutation on HLMVECs edited by CRISPR-Cas9 with Tie2 sgRNAs as showing a series of bands. Wild-types cells with or without Cas9 or vector only show single bands (negative controls).

    Journal: Scientific Reports

    Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

    doi: 10.1038/srep42127

    Figure Lengend Snippet: Deletion of Tie2 gene in primary ECs using CRISPR-Cas9. ( A ) Cultured primary HLMVECs were transduced by EGFP-Cas9 adenovirus and sgRNA lentivirus targeting on Tie2 and subjected to flow cytometry analysis of EGFP expression. In a separate study, HLMVECs were transduced by GFP lentivirus and subjected to flow cytometry analysis of EGFP expression as an indicator of lentivirus transduction efficiency. ( B ) Protein expression of Tie2 in vector control and Tie2 knockout HLMVECs induced by CRISPR-Cas9 was determined by immunoblotting. Overexpression of mouse Tie2 in Tie2 −/− HLMVECs was determined to assess the ability to rescue Tie2 expression after deletion. The uncropped full-length gels can be found in Supplementary Fig. S1 . ( C ) Quantification of Tie2 protein expression from 3 independent experiments. sgRNA Tie2-1 and sgRNA Tie2-2 are CRISPR-Cas9-mediated deletions of Tie-2 at two distinction domains of Tie2 (two different sgRNAs). HuTie2KO1 and HuTie2KO2 + mTie2 represent restoration of Tie-2 expression in cells having undergone Tie-2 deletion. Differences were calculated using one-way ANOVA. P values less than 0.05 are indicated in the graph. ( D ) T7E1 assay detecting mutation on HLMVECs edited by CRISPR-Cas9 with Tie2 sgRNAs as showing a series of bands. Wild-types cells with or without Cas9 or vector only show single bands (negative controls).

    Article Snippet: Rescue of Tie2 expression in HLMVECs following Tie-2 deletion Mouse Tie2 (mTie2) cDNA plasmids was purchased from Sino Biological Inc. (# MG51087-G).

    Techniques: CRISPR, Cell Culture, Flow Cytometry, Expressing, Transduction, Plasmid Preparation, Knock-Out, Over Expression, Mutagenesis

    Tie2 deletion by CRISPR-Cas9 in primary ECs increases endothelial permeability and mitigates recovery of permeability in response to thrombin challenge. ( A ) Basal TER and post-thrombin (1 U/ml) TER were studied in confluent control, Tie2-deleted HLMVECs and mTie2 overexpressing cells in which Tie2 had been deleted. Absolute TER values were reduced in both Tie2-deleted groups as compared to control ECs at basal condition. mTie2 overexpression successfully rescued the basal leakiness. ( B ) Quantification of TER values of wild-type (control), transduced cells (sgRNA Tie2-1, sgRNA Tie2-2) and rescued cells (HuTie2KO + mTie2) at basal (−1 h), thrombin-stimulated (0.5 h) and post-recovery (3 h) condition. Differences were calculated using two-way ANOVA. P values less than 0.05 are indicated in the graph. n = 3. ( C ) Serum-starved confluent control or Tie2-deleted HLMVECs were challenged by PBS or 1 U/ml of thrombin, and subjected for VE-cadherin immunostaining at the indicated time-points and analyzed by confocal microscopy. The marked disruption of VE-cadherin junctions seen in wild-type HLMVEC monolayer (control) at the 30 min post thrombin (white arrows) was reversed by 2 h; however, the defective VE-cadherin junctions were present in Tie2-deleted HLMVECs 2 h post-thrombin. White arrows are used to identify areas of adherens junction disruption where neighboring cells lack cell membrane localization of VE-cadherin. Results are representative of 3 independent experiments.

    Journal: Scientific Reports

    Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

    doi: 10.1038/srep42127

    Figure Lengend Snippet: Tie2 deletion by CRISPR-Cas9 in primary ECs increases endothelial permeability and mitigates recovery of permeability in response to thrombin challenge. ( A ) Basal TER and post-thrombin (1 U/ml) TER were studied in confluent control, Tie2-deleted HLMVECs and mTie2 overexpressing cells in which Tie2 had been deleted. Absolute TER values were reduced in both Tie2-deleted groups as compared to control ECs at basal condition. mTie2 overexpression successfully rescued the basal leakiness. ( B ) Quantification of TER values of wild-type (control), transduced cells (sgRNA Tie2-1, sgRNA Tie2-2) and rescued cells (HuTie2KO + mTie2) at basal (−1 h), thrombin-stimulated (0.5 h) and post-recovery (3 h) condition. Differences were calculated using two-way ANOVA. P values less than 0.05 are indicated in the graph. n = 3. ( C ) Serum-starved confluent control or Tie2-deleted HLMVECs were challenged by PBS or 1 U/ml of thrombin, and subjected for VE-cadherin immunostaining at the indicated time-points and analyzed by confocal microscopy. The marked disruption of VE-cadherin junctions seen in wild-type HLMVEC monolayer (control) at the 30 min post thrombin (white arrows) was reversed by 2 h; however, the defective VE-cadherin junctions were present in Tie2-deleted HLMVECs 2 h post-thrombin. White arrows are used to identify areas of adherens junction disruption where neighboring cells lack cell membrane localization of VE-cadherin. Results are representative of 3 independent experiments.

    Article Snippet: Rescue of Tie2 expression in HLMVECs following Tie-2 deletion Mouse Tie2 (mTie2) cDNA plasmids was purchased from Sino Biological Inc. (# MG51087-G).

    Techniques: CRISPR, Permeability, Over Expression, Immunostaining, Confocal Microscopy