Construction of macrophage RAW 264.7 cells with gsdmd gene knockout by CRISPR/Cas9 system

To construct a cell model of gene knockout in macrophage RAW 264.7 cells using CRISPR/Cas9 system. Four specific single guide RNAs (sgRNAs) targeting were designed to construct pGL3-sgRNA recombinant plasmids, which were identified by PCR amplification and sequencing.Cas9 and the recombinant plasmid...

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Bibliographic Details
Published in:Nan fang yi ke da xue xue bao = Journal of Southern Medical University 2021-01, Vol.41 (1), p.116-122
Main Authors: Zhou, Liting, Ye, Ying, Yuan, Haibo, Wu, Chaoyi, Wu, Shuyan
Format: Article
Language:Chinese
Subjects:
Animals
CRISPR-Cas Systems - genetics
Gene Knockout Techniques
Mice
Plasmids
RAW 264.7 Cells
RNA, Guide, CRISPR-Cas Systems
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Summary:To construct a cell model of gene knockout in macrophage RAW 264.7 cells using CRISPR/Cas9 system. Four specific single guide RNAs (sgRNAs) targeting were designed to construct pGL3-sgRNA recombinant plasmids, which were identified by PCR amplification and sequencing.Cas9 and the recombinant plasmids were transfected into RAW 264.7 cells in two steps, and the cellular expression of was detected with real-time quantitative PCR (qPCR).The positive cell clones with gene knockout were screened using puromycin and verified by sequencing and Western blotting.Annexin Ⅴ/PI staining and LDH release assay were performed in -/-RAW 264.7 cells after being co-cultured with . qPCR results showed that gene was stably expressed in RAW 264.7-Cas9 cells ( < 0.01).PCR and sequencing results demonstrated successful construction of the recombinant plasmid pGL3-sgRNA. The results of PCR, sequencing and Western blotting all confirmed that RAW 264.7 cells were successfully constructed. Annexin Ⅴ/PI staining and LDH release assay sho
ISSN:1673-4254
DOI:10.12122/j.issn.1673-4254.2021.01.17