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

作　　者：陈健康[1,2] 魏从文[3] 梁慧[2] 王贝晗[2] 钟辉[3] 杨晓莉[1,2] 

机构地区：[1]安徽医科大学武警总医院临床学院,合肥230032 [2]武警总医院,北京100039 [3]军事医学科学院生物工程研究所,北京100850

出　　处：《军事医学》2016年第7期549-553,共5页Military Medical Sciences

基　　金：武警部队院级二类课题资助项目(WZ 2010016;WZ2011021)

摘　　要：目的利用CRISPR/Cas9基因编辑系统敲除小鼠源肝癌细胞H22中的GP73基因,构建H22细胞GP73基因敲除的稳定细胞株。方法根据CRISPR/Cas9靶点设计原则,设计2条特异性识别GP73基因启动子的上下游sgRNA,利用载体p X459质粒,构建2对重组真核表达质粒。经酶切和测序鉴定后,将重组质粒转染至H22细胞内,使用嘌罗霉素加压筛选稳定敲除GP73的H22细胞株,利用免疫印迹检测重组质粒对内源GP73的敲除效果。MTT实验检测GP73被敲除后对细胞增殖能力的影响,再利用划痕实验检测细胞的迁移能力。结果免疫印迹结果说明敲除GP73基因的小鼠源H22细胞株内无GP73蛋白的表达;并且GP73被敲除后,H22细胞的增殖能力和迁移能力减慢。结论通过CRISPR/Cas9系统获得了靶向GP73基因的重组质粒,并且筛选出了稳定干扰GP73表达的细胞株,从而为探讨GP73在肝癌发生中的作用奠定基础。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 GP73 gene first promoter were designed before a recombinant eukaryotic expressional plasmid was constructed using p X459. 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 GP73 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 GP73 in hepatocarcinogenesis.

关 键 词：CRISPR/Cas9系统 GP73 基因敲除 癌 肝细胞 

分 类 号：R735.7[医药卫生—肿瘤]