基于CRISPR/Cas9技术构建PKCα基因敲除的人腹膜间皮细胞系  

Construction of human peritoneal mesothelial cell lines with PKCαgene knockout with the technique of CRISPR/Cas9

作　　者：吴忠财 马祖福 梁望群 左学志 姚颖[1] 徐钢[1] 王艻 Wu Zhong-cai;Ma Zu-fu;Liang Wang-qun;Zuo Xue-zhi;Yao Ying;Xu Gang;Wang Le(Division of Nephrology,Affiliated Tongji Hospital,Tongji Medical College,Huazhong University of Science&Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院肾病内科,武汉430030

出　　处：《临床肾脏病杂志》2021年第9期750-754,共5页Journal Of Clinical Nephrology

基　　金：国家自然科学基金(81873630)。

摘　　要：目的通过CRISPR/Cas9技术敲除人腹膜间皮细胞系HMrSV5的PKCα基因,为腹膜纤维化的发病机制研究提供体外细胞模型。方法针对人PKCα基因外显子区域设计3对sgRNA序列,其中两对序列靶点位于第4外显子,一对序列靶点位于第3外显子。由引物合成公司合成两端含酶切位点黏端的寡聚双链DNA,酶切连接至真核表达载体GV393。使用293T细胞进行慢病毒包装,通过病毒感染HMrSV5细胞,检测HMrSV5细胞中PKCα的敲除效率即脱靶情况。结果3对sgRNA通过慢病毒表达载体成功转染HMrSV5细胞,经实时荧光定量PCR及免疫印迹检测两对sgRNA成功敲低PKCα。错配酶和基因测序结果显示PKCα基因组DNA导入有效突变,3个脱靶位点与原参考序列一致,排除脱靶情况。结论利用CRISPR/Cas9技术成功构建了PKCα敲除的HMrSV5细胞,有利于相关基因体外研究。Objective To construct an in vitro cell model for elucidating the pathogenesis of peritoneal fibrosis by a knockout of PKCαgene in human peritoneal mesothelial cell line HMRSV5 with the technique of CRISPR/Cas9.Methods Three pairs of sgRNA sequences were designed for exon region of human PKCαgene.Two pairs of sequences were targeted at exon 4 and one pair exon 3.The oligomeric double-stranded DNA was synthesized by a primer synthetic company.The oligomeric double-stranded DNA was digested and ligated to eukaryotic expression vector GV393.The knockout efficiency of PKCα,i.e.off-target condition,was detected by using 293T cells as lentivirus packaging.Results Three pairs of sgRNA were successfully transfected into HMRSV5 cells by lentiviral expression vector.Real-time quantitative polymerase chain reaction(PCR)and Western blot revealed that PKCαwas successfully knocked down in two pairs of sgRNA.The results of mismatch enzyme and gene sequencing indicated that PKCαgenome DNA was effectively mutated and three off-target sites were consistent with the original reference sequence,excluding off-target condition.Conclusion The PKCαknockout HMRSV5 cells have been successfully constructed through the technique of CRISPR/Cas9.It may benefit further in vitro studies of related genes.

关 键 词：腹膜纤维化 PKCΑ CRISPR/Cas9 基因敲除 

分 类 号：R459.5[医药卫生—治疗学]