基于CRISPR/Cas9系统构建Slfn2基因敲除的小鼠肺癌细胞系  

Construction of the Slfn2 Knockout Cell Lines in Lewis Lung Carcinoma Model Based on CRISPR/Cas9 System

作　　者：陈莹[1,2] 周祖平[1,2,3] 邢兵[1] 蒲仕明 CHEN Ying;ZHOU Zuping;XING Bing;PU Shiming(College of Life Sciences,Guangxi Normal University,Guilin Guangxi 541006,China;Guangxi Universities Key Laboratory of Stem Cell and Biopharmaceutical Technology(Guangxi Normal University),Guilin Guangxi 541004,China;Biomedical Research Center of Guangxi Normal University,Guilin Guangxi 541004,China)

机构地区：[1]广西师范大学生命科学学院,广西桂林541006 [2]广西高校干细胞与医药生物技术重点实验室(广西师范大学),广西桂林541004 [3]广西师范大学生物医学研究中心,广西桂林541004

出　　处：《广西师范大学学报（自然科学版）》2022年第4期173-179,共7页Journal of Guangxi Normal University:Natural Science Edition

基　　金：国家自然科学基金(81972700,61827819);广西自然科学基金(2018GXNSFBA281115);广西大学生创新创业训练计划(202010602052)。

摘　　要：为构建Slfn2^(-/-)小鼠肺癌细胞系(Lewis lung carcinoma,LLC),本文利用CRISPR/Cas9技术,在CHOPCHOP网站筛选Slfn2基因的sgRNA序列,将合成的oligo序列退火后连接至pLenti CRISPR v2质粒中,并与pMD2.G、psPAX2共转染至293T细胞中包装慢病毒;利用包装的慢病毒感染LLC细胞,经筛选和单克隆培养,获取Slfn2^(-/-)敲除的LLC细胞。结果显示:SgRNA成功插入载体重组质粒;利用3质粒包装的慢病毒成功将LLC中的Slfn2基因突变,获得丢失8个碱基移码突变和丢失180个碱基缺失突变的Slfn2^(-/-)小鼠肺癌细胞。可见,利用CRISPR/Cas9技术成功构建了Slfn2基因敲除的小鼠肺癌细胞。To construct a stable knocking down of Slfn2 expression cell line in Lewis lung carcinoma(LLC)using CRISPR/Cas9 system,a guide RNA(sgRNA)sequence targeting the Slfn2 gene was designed by using the CHOPCHOP website software.After ligation of the sgRNA to the lenti-CRISPR-v2 plasmid,the T293 cells were co-transfected with pLenti CRISPR v2-Slfn2,pMD2.G and psPAX2 to generate Lentivirus.The packaged lentivirus was used to infect LLC cells,and Slfn2^(-/-)knockout LLC cells were obtained by screening and Monoclone culture.The results showed that the two SgRNAs were successfully inserted into the vector respectively.Slfn2 gene in LLC was successfully mutated by lentivirus packaged with three plasmids.Two Slfn2 knockout LLC cell lines were obtained,one with 8 bp deletion plus frameshift mutation and the other with 180 bp deletion mutation.The mouse lung cancer cells knocked out by Slfn2 gene were successfully constructed by CRISPR/Cas9 technology.

关 键 词：肺癌 慢病毒 Slfn2 CRISPR/Cas9 

分 类 号：R734.2[医药卫生—肿瘤]