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Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR.

ABSTRACT: Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from puromycin-selected ES cells.

SUBMITTER: Liu Y

PROVIDER: S-EPMC9184805 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR.

Liu Yuhan Y Chen Qian Q Song Chenglin C Xu Zhen Z Yang Shuting S Li Xiajun X

STAR protocols 20220607 2

Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from pur ...[more]

PMID: 35693210

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