利用CRISPR/Cas9技术构建CYP2E1基因敲除的HEK293FT细胞系  

Construction of CRISPR/Cas9-mediated genomic deletion of the CYP2E1 gene in human HEK293FT cell line

作　　者：范启明[1] 李若碧 王飞[1] 郭涛[1] 王婷[1] 李道传[1] 邢秀梅[1] 陈丽萍[1] 陈雯[1] 王庆[1] FAN Qiming;LI Ruobi;WANG Fei;GUO Tao;WANG Ting;LI Daochuan;XING Xiumei;CHEN Liping;CHEN Wen;WANG Qing(School of Public Health, Sun Yat-sen University,Guangzhou 510080, Guangdong, China)

机构地区：[1]中山大学公共卫生学院,广东广州510080

出　　处：《癌变．畸变．突变》2017年第5期352-357,363,共7页Carcinogenesis,Teratogenesis & Mutagenesis

摘　　要：目的:利用CRISPR/Cas9系统在HEK293FT细胞系中敲除CYP2E1基因并探讨其在检测化学物毒性中的应用。方法:设计3对分别靶向CYP2E1基因第2、第3和第7外显子的sg RNA序列并构建PX461表达载体;将携带靶向CYP2E1的sg RNA表达载体转染至HEK293FT细胞中,通过SURVEYOR检测分析sg RNA的切割效率;利用有限稀释法获得CYP2E1敲除细胞株,通过实时荧光定量PCR和蛋白印迹检测细胞株中CYP2E1的敲除效果;并检测其对N-(4-羟基苯基)乙酰胺和1,2-二氯乙烷的细胞毒性效应的影响。结果:SURVEYOR检测分析靶向CYP2E1基因第3外显子的sg RNA的切割效率为23%;成功构建了2株CYP2E1基因敲除的细胞株,实时荧光定量PCR结果显示CYP2E1 m RNA表达下降,蛋白印迹结果显示CYP2E1蛋白无表达;CYP2E1基因敲除会显著降低N-(4-羟基苯基)乙酰胺和1,2-二氯乙烷的细胞毒性。结论:通过CRISPR/Cas9构建出CYP2E1稳定敲除的肾细胞系,为研究CYP2E1的功能和作用机制提供了有效的工具。OBJECTIVE:To construct the CYP2E1knock-out(KO)HEK293FT cell line using CRISPR/Cas9system and explore its usefulness in chemical toxicity testing.METHODS:The sgRNA expression vector targetingCYP2E1gene was transfected into HEK293FT cells.Single cells from the transfected cells were isolated through serialdilutions to get the HEK293FT CYP2E1KO cell line.The cell line was tested for expression of cytotoxicity from N-(4-hydroxyphenyl)-acetamide(4-APAP)and1,2-dichloroethane(1,2-DCE).RESULTS:Two HEK293FT CYP2E1KO celllines were obtained and they showed dramatic reduction in susceptibility to N-(4-hydroxyphenyl)-acetamide(4-APAP)and1,2-dichloroethane(1,2-DCE)cytotoxicity.CONCLUSION:HEK293FT CYP2E1KO cell lines were successfullyconstructed using a CRISPR/Cas9system and they can be useful for studying the function of CYP2E1.

关 键 词：CYP2E1 CRISPR/Cas9 基因敲除 HEK293FT细胞系 

分 类 号：R392.11[医药卫生—免疫学]