敲除SOSSB1、SOSSB2和SOSSC基因稳定HeLa细胞株的构建  

Construction of HeLa cell lines with SOSSB1,SOSSB2 and SOSSC genes knocked out

作　　者：孙瑜 陈红霞[2] 齐永[2] 李素芹[2] 沈万鹏 饶继先 李佳萌[2] 李晓玲 李越希[1,2,3] SUN Yu;CHEN Hong-xia;QI Yong;LI Su-qin;SHEN Wan-peng;RAO Ji-xian;LI Jia-meng;LI Xiao-ling;LI Yue-xi(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Nanjing Medical University,Nanjing 210029,Jiangsu Province,China)

机构地区：[1]南京医科大学基础医学院生物化学与分子生物学系,江苏南京210029 [2]南京军区军事医学研究所,江苏南京210002 [3]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《中国生物制品学杂志》2018年第11期1197-1200,1206,共5页Chinese Journal of Biologicals

基　　金：江苏省自然青年基金项目(BK20160131;BK20160130);国家自然青年基金项目(31701181;31600151)

摘　　要：目的构建SOSSB1、SOSSB2和SOSSC基因敲除的稳定HeLa细胞株。方法分别设计并合成SOSSB1、SOSSB2和SOSSC亚基的两对靶向基因特定外显子的A、B两个单导向核酸(single guide RNA,sgRNA)及其互补链,与含BbsⅠ黏性末端的PX462载体连接,构建重组质粒。重组质粒经酶切和测序鉴定正确后,分别转染至HeLa细胞,用2μg/mL的嘌呤霉素培养筛选出稳定HeLa细胞。采用Western blot法对敲除效果进行鉴定。结果 A、B sgRNA均成功插入至PX462载体,重组质粒经酶切和测序鉴定,证明构建正确。Western blot结果表明,筛选出的SOSSB1、SOSSB2和SOSSC基因敲除稳定HeLa细胞株分别不表达SOSSB1、SOSSB2、SOSSC蛋白。结论成功构建了SOSSB1、SOSSB2和SOSSC基因敲除的稳定HeLa细胞株。Objective To construct the stable HeLa cell lines with SOSSB1,SOSSB2 and SOSSC genes knocked out.Methods The A and B sgRNAs（single guide RNA）targeting to exons of SOSSB1,SOSSB2 and SOSSC genes and the complementary strands were designed and synthesized,then inserted into Bbs Ⅰ cohesive ends-containing PX462 linear vector. The constructed recombinant plasmids were identified by restriction analysis and sequencing and transfected to HeLa cells. The stable HeLa cells were screened with 2 μg/mL puromycin. The result of knockout was identified by Western blot. Results Both A and B sgRNAs were successfully inserted into PX462 vector. Restriction analysis and sequencing proved that recombinant plasmids were constructed correctly. Western blot showed no expressions of SOSSB1,SOSSB2 and SOSSC proteins in HeLa cells with the corresponding genes knocked out. Conclusion Stable HeLa cell lines with SOSSB1,SOSSB2 and SOSSC genes knocked out were successfully constructed.

关 键 词：SOSS基因 基因敲除 HELA细胞株 CRISPR/Cas9n双切口酶 

分 类 号：Q28[生物学—细胞生物学]