军事医学2016，Vol.40Issue(7)：549-553,5.DOI:10.7644/j.issn.1674-9960.2016.07.004
利用CRISPR／Cas9系统构建H22细胞GP73基因敲除稳定株及功能鉴定
Construction of H22 GP73 knockout gene stable strain using CRISPR/Cas9 gene editing system and identification of functions
摘要
Abstract
Objective To knock out the GP73 gene in H22 cells originating in mice using CRISPR/Cas9 gene editing system and construct H22 GP73 gene knockout stable strain for identification of its functions .Methods Two pairs of sgRNAs that could specifically identify the upstream and downstream of GP 73 gene first promoter were designed before a recombinant eukaryotic expressional plasmid was constructed using pX 459 .After enzyme digestion and sequencing , two pairs of recombinant plasmids were co-transfected into H22 cells before puromycin was used to screen positive cells and monoclonal cells which stably knocked out GP 73 gene were developed .The knockout effect was measured by Western blotting.Cell Titer 96® AQueous One Solution Assay was used to detect the effect on cell reproductive capacity when the GP73 was knocked out .The transferability was detected through wound healing test .Results The result of Western blotting suggested that GP73 protein was undetected in the construction of H22 GP73 knockout gene stable strain after transfection.The transfer and reproduction slowed down .Conclusion H22 GP73 gene knockout stable strain can be successfully built using CRISPR/Cas9 gene editing system ,thus facilitating studies on the function of GP 73 in hepatocarcinogenesis .关键词
CRISPR/Cas9系统/GP73/基因敲除/癌,肝细胞Key words
CRISPR/Cas9/GP73/gene knockout/carcinoma ,hepatocellular分类
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陈健康,魏从文,梁慧,王贝晗,钟辉,杨晓莉..利用CRISPR／Cas9系统构建H22细胞GP73基因敲除稳定株及功能鉴定[J].军事医学,2016,40(7):549-553,5.基金项目
武警部队院级二类课题资助项目 （）
