利用CRISPR/Cas9系统在小鼠胚胎干细胞中进行miR-22的功能研究

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

旨在构造neomycin筛选标记的sgRNA表达载体,并利用CRISPR/Cas9技术构建miR-22缺失的小鼠胚胎干细胞,探究miR-22在小鼠胚胎干细胞中的调控作用。首先通过点突变、搭桥PCR等手段,构造neomycin筛选标记的sgRNA表达载体,并获得靶向敲除miR-22的sgRNA表达载体,电转至稳转Cas9的小鼠胚胎干细胞中;其次经药物筛选、基因型鉴定等步骤筛选miR-22纯合缺失的小鼠胚胎干细胞。RT-qPCR手段证实miR-22在小鼠胚胎干细胞中被成功敲除,纯合突变体的比例约为6.67%。此外,miR-22缺失并未影响胚胎干细胞的细胞形态以及Oct4、Sox2和Nanog等干性基因的表达。因此,miR-22对小鼠胚胎干细胞的干性维持并非必需,而对胚胎干细胞谱系分化和命运决定的影响还有待进一步研究。

This study aims to construct neomycin-resistant sgRNA expression plasmids and to generate miR-22 knockout mouse embryonic stem cells（m ESCs）with CRISPR/Cas9 for examining miR-22＇s regulatory roles. First,point mutation and overlap PCRwere performed to construct neomycin-resistant sgRNA expression plasmids. Then,sgRNA expression plasmids of miR-22 knockout wereelectroporated into m ESCs with stable Cas9 expression. After neomycin selection and PCR genotyping,the miR-22 homozygous knockoutm ESCs 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 m ESC as well as the expressions of pluripotentmarkers 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 m ESC differentiation and cell fate decision need further identification.