Functional Study of miR-22 in Mouse Embryonic Stem Cell with CRISPR/Cas9 System

doi:10.13560/j.cnki.biotech.bull.1985.2017-0964

Abstract

Abstract: This study aims to construct neomycin-resistant sgRNA expression plasmids and to generate miR-22 knockout mouse embryonic stem cells（mESCs）with CRISPR/Cas9 for examining miR-22’s regulatory roles. First,point mutation and overlap PCR were performed to construct neomycin-resistant sgRNA expression plasmids. Then,sgRNA expression plasmids of miR-22 knockout were electroporated into mESCs with stable Cas9 expression. After neomycin selection and PCR genotyping,the miR-22 homozygous knockout mESCs were screened. The homozygous deletion was confirmed by RT-qPCR experiments and the efficiency of homozygous knockout was about 6.67%. In addition,the deletion of miR-22 presented no obvious effect on the morphology of mESC as well as the expressions of pluripotent markers including Oct4,Sox2 and Nanog. Together,miR-22 may be not necessarily required for the maintenance of embryonic stem cell,while the roles of miR-22 in controlling mESC differentiation and cell fate decision need further identification.

Key words: mouse embryonic stem cell, CRISPR/Cas9, miR-22

ZHANG Xue, CHEN Liang-liang, DAI Hong-xia, ZHANG Wen-sheng, REN Wen-yan. Functional Study of miR-22 in Mouse Embryonic Stem Cell with CRISPR/Cas9 System[J]. Biotechnology Bulletin, 2018, 34(5): 71-79.

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