利用基于CRISPR-Cas9的基因编辑技术对miR-29a在GT1-7细胞中进行基因敲除  

The Gene Knockout of miR-29a in GT1-7 Cells based on Gene Editing by CRISPR-Cas9 System

作　　者：王斯佳[1] 李晓宁[1] 李文文[1] 李凯[1] 肖君华[1] 周宇荀[1] 

机构地区：[1]东华大学生物科学与技术研究所,上海201620

出　　处：《现代生物医学进展》2016年第21期4028-4031,4041,共5页Progress in Modern Biomedicine

基　　金：中央高校基本科研业务专项资金(2232013A3-06)

摘　　要：目的:本文利用CRISPR-Cas9技术在GT1-7细胞中对miR-29a基因进行基因编辑,用于构建miR-29a基因敲除GT1-7细胞模型。方法:通过构建Cas9稳转的GT1-7细胞株并转染sgRNA质粒用于在靶向miR-29a基因区域引发突变。然后构建EGFP与sgRNA共表达质粒并转染Cas9稳转GT1-7细胞,利用流式细胞仪富集表达绿色荧光蛋白的阳性细胞和分选阳性单克隆细胞。最后利用实时荧光定量PCR(realtimefluorescencequantitativePCR)对富集细胞和单克隆细胞进行miR-29a表达量检测。结果:T7E1检测结果显示CRISPR-Cas9系统有效地在miR-29a基因区域引发了突变。荧光定量PCR结果显示,与对照组相比,富集后阳性细胞miR-29a的表达量整体下降了50%左右(P<0.05)。此外,通过流式筛选获得了一个纯合miR-29a基因敲除细胞克隆,与对照组相比,其miR-29a的表达量下降了75%左右(P<0.05)。结论:本文建立了一种有效编辑GT1-7细胞基因的方法,并采用该方法构建了miR-29a稳定敲除细胞模型。Objective： CRISPR-Cas9 technology was used to edit mi R-29 a gene in GT1-7 cells and construct mi R-29 a knockout GT1-7 cell model. Methods： To induce mutation on mi R-29 a loci, Cas9 steadily expressed GT1-7 cells were constructed and transfected by sg RNA plasmids. Then a plasmid co-expressing EGFP and sg RNA was constructed and transfected into Cas9 expressing cells, flow cytometry was applied to enrich EGFP positive cells and test positive Monoclonal cell. Finally RT-PCR was used to detect the expression levels of mi R-29 a in enriched cells and Monoclonal cell. Results： Mutation could be efficiently induced on mi R-29 a loci by CRISPR-Cas9 system detected by T7E1 assays. Compared with control group, the expression of mi R-29 a in enriched positive cells totally decreased by 50 percent（P〈0.05） using RT-PCR. Furthermore, one homogeneous mi R-29 a gene knockout cell clone was obtained by flow screening. The mi R-29 a expression of this cell clone decreased by 75 percent（P〈0.05）compared with control group. Conclusions：An effective gene edit- ing method in GT1-7 cells was established, which constructed a mi R-29 a steadily knockout cell model.

关 键 词：CRISPR-Cas9 GT1-7 Mir-29a 单克隆细胞 

分 类 号：Q75[生物学—分子生物学] Q78