CRISPR/Cas9技术制备猪肌肉生长抑制素基因敲除细胞

Preparation of Myostatin Gene Knock Out Pig Cells Using a CRISPR/Cas9 System

采用CRISPR/Cas9技术制备质粒HRX-2MCS和PX330,两种质粒转染湖北白猪胎儿成纤维细胞,G418筛选抗性细胞株。收集培养细胞提取总DNA,分子定位PCR法检测绿色荧光蛋白(GFP)基因在猪肌肉生长抑制素(MSTN)基因外显子上的定位整合情况,得到T1靶位点整合细胞5株和T3靶位点整合细胞3株。Q-PCR定量分析结果表明3108号阳性细胞株MSTN基因m RNA表达量减少50%,实验获得的基因敲除细胞可以用于体细胞核移植法制备MSTN基因敲除猪的研究。

These plasmids HRX-2MCS and PX330 were prepared using a CRISPR/Cas9 system, and two plasmids were transfected into Hubei white pig fetal fibroblasts with G418 screening resistant cell lines. Collecting culture cells and extracting the total DNA, the integration sites of the green fluorescent protein(GFP) gene were detected,which located in pig myostatin(MSTN) gene exon by molecular location PCR. This test got 5 cell strains which had T1 target site integration of GFP gene and 3 cell strains which had T3 target site integration of GFP gene.Q-PCR quantifative analysis results showed that m RNA expression amount of MSTN gene decreased by 50% in the positive cell line 3108, and this knock out cells can be used for preparation of MSTN gene knock out pigs by somatic cell nuclear transfer.