基于CRISPR/Cas9技术构建与鉴定敲除ACE2基因的Huh7肝癌细胞株  

Construction and Identification of Huh7 Hepatoma Cell Line with ACE2 Gene Knockout Based on CRISPR/Cas9 Technology

作　　者：张祖霖 刘方芳 周青鸟 赵瑞强 贺菽嘉 林文珍 ZHANG Zu-lin;LIU Fang-fang;ZHOU Qing-niao;ZHAO Rui-qiang;HE Shu-jia;LIN Wen-zhen(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Guangxi Medical University,Nanning 530021;Key Laboratory of Biological Molecular Medicine Research(Guangxi Medical University),Education Department of Guangxi Zhuang Autonomous Region,Nanning 530021)

机构地区：[1]广西医科大学基础医学院生物化学与分子生物学教研室,南宁530021 [2]广西高校生物分子医学研究重点实验室,南宁530021

出　　处：《生物技术通报》2023年第6期181-188,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(81660464)。

摘　　要：利用CRISPR-Cas9基因编辑技术敲除人Huh7肝癌细胞的血管紧张素转化酶2(ACE2)基因,构建ACE2基因敲除细胞株,为研究ACE2在肝细胞癌的作用提供细胞模型。首先,对ACE2结构域进行鉴定,利用在线网站设计两条破坏所有结构域、靶向作用于ACE2外显子的sgRNA。其次,构建重组载体并转染肝癌细胞Huh7,嘌呤霉素筛选出单克隆细胞株。最后,免疫印迹鉴定敲除效果。结构域鉴定结果显示,在340-520号氨基酸位置存在Zn结合位点和激活位点,根据sgRNA靶点设计原则,采用片段敲除的方式,针对ACE2的第9、第10外显子设计两对sgRNA,并成功构建PX459-ACE2-sgRNA重组质粒。嘌呤霉素成功筛选出单克隆细胞株,并测序证实了发生片段敲除,ACE2蛋白在敲除细胞株中不表达;成功构建ACE2敲除的Huh7细胞株,为日后研究ACE2在肝细胞癌的发生机制奠定基础。The experiment is aimed to establish ACE2(angiotensin converting enzyme 2)-knock out Huh7 liver cancer cells using CRISPR-Cas9,which could be used as cell model to study the role of ACE2 in HCC(hepatocellular carcinoma).Firstly,the ACE2 domain was identified,and two sgRNAs targeting ACE2 exons were designed using an online website that destroyed all the domains.Secondly,the recombinant vector was constructed and transfected into HCC cell Huh7,and the monoclonal cell line was screened by puromycin.Finally,the knockout effect was identified by immunoblotting.The domain identification results showed that there were Zn-binding sites and activation sites at amino acids 340-520.According to the sgRNA target design principle,two pairs of sgRNA were designed for the ninth and tenth exons of ACE2 by fragment knockout method,and the recombinant plasmid PX459-ACE2-sgRNA was successfully constructed.The monoclonal cell line was successfully screened by puromycin,sequencing confirmed the occurrence of fragment knockout,and ACE2 protein was not expressed in the ACE2 knockout cell line.CRISPR-Cas9 technology was used to successfully construct ACE2 knockout Huh7 cell line,which lays a foundation for future research on the mechanism of ACE2 in HCC.

关 键 词：血管紧张素转化酶2 肝细胞癌 CRISPR-Cas9 单克隆细胞株 

分 类 号：R735.7[医药卫生—肿瘤]