通过CRISPR-Cas9系统敲减PAEC中GGTA1和CMAH基因的效果检测

The Detection of the Knocked-down Effects of GGTA1 and CMAH in PAEC by CRISPR-Cas9 System

目的:检测由短回文重复序列(CRISPR)-Cas9系统敲减猪动脉内皮细胞(PAEC)中基因α1,3半乳糖基转移酶(GGTA1)和CMP-N-乙酰神经氨酸羟化酶(CMAH)的效果。方法:通过CRISPR-Cas9系统,用已设计好的GGTA1和CMAH基因的guide RNA(g RNA)构建质粒,以慢病毒为载体包装病毒和感染PAEC,对成功感染的细胞进行单克隆培养并挑选收集单克隆细胞,提取细胞基因组,进行PCR,琼脂糖凝胶电泳,退火、酶切,测序等方法对GGTA1和CMAH基因敲减的效果进行检测。结果:对单克隆细胞进行选择性测序,选择V2-g-a1、V2-c-b1、V2-c-d4单克隆细胞测序结果分别为:V2-g-a1细胞基因组突变位点在第205位;V2-c-b1细胞基因组突变位点在第240位;V2-c-d4细胞基因组突变位点在第246位。结论:CRISPR-Cas9系统成功构建质粒,完成慢病毒包装和感染细胞,成功敲减目的基因。

Objective The detection of the knocked-down effects of GGTA1 and CMAH in PAEC by CRISPR-Cas9 system. Methods using Lentiviral as carrier to packaged virus which to infect the PAEC（The pig artery endothelial cells）, choosing the successfully infected cell to monoclonal culture and collect monoclonal cell, extracting cell＇s genome, conducting PCR, agarose gel electrophoresis, annealing, digestion and sequencing methods to detection of the knocked-down effects of GGTA1 and CMAH. Results Selective sequencing monoclonal cell and selecting V2-g-a1, V2-c-b1, V2-c-d4 cell to sequencing. The results show that V2-g-a1 cell genome mutate in the 205th; V2-c-b1 cell genome mutate in the 240th; V2-c-d4 cell genome mutate in the 246 th. Conclusions CRISPR-Cas9 system has been successfully constructed plasmids, accomplished lentiviral packaged and infected cells, target genes are knocked-down successfully.