基于CRISPR/Cas9技术的SS18-SSX1阳性细胞模型的构建与验证  

Construction and validation of SS18-SSX1 fusion gene positive cell model based on CRISPR/Cas9 technology

作　　者：田娇 张乾儒 王宁[1] 史奇 李亚 张琨岚 李锋[1,2] TIAN Jiao;ZHANG Qianru;WANG Ning;SHI Qi;LI Ya;ZHANG Kunlan;LI Feng(Department of Pathology and Key Laboratory for Xinjiang Endemic and Ethnic Diseases,School of Medicine,Shihezi University,Shihezi,Xinjiang 832000,China;Department of Pathology and Medical Research Center,Beijing Chaoyang Hospital,Capital Medical University,Beijing 100020,China)

机构地区：[1]石河子大学医学院,新疆石河子832000 [2]首都医科大学附属北京朝阳医院医学研究中心,北京100200

出　　处：《石河子大学学报(自然科学版)》2025年第1期8-15,共8页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(82173057,82002846)。

摘　　要：目的 利用CRISPR/Cas9技术构建并验证SS18-SSX1^(+)-HEK293T细胞模型。方法 靶向SS18和SSX1分别设计sgRNA,构建Lentivirus-CRISPR v2-SS18-gRNA-SSX1-gRNA-EGFP一体化慢病毒载体,通过慢病毒转染和荧光流式分选技术筛选转入载体的HEK293T多克隆细胞,在多克隆细胞的基础上挑选SS18-SSX1阳性的单克隆细胞株,通过RT-PCR、Sanger测序和Western Blot验证SS18-SSX1^(+)-HEK293T单克隆细胞系构建成功。结果 成功构建了Lentivirus-CRISPR v2-SS18-gRNA-SSX1-gRNA-EGFP慢病毒载体;通过RT-PCR、Sanger测序和WB实验在178个HEK293T单克隆细胞中成功筛选并验证出1株SS18-SSX1阳性的单克隆细胞系。结论 利用CRISPR/Cas9技术成功构建SS18-SSX1融合基因阳性的HEK293T细胞系。Objective The construction and validation of a cell model utilising the CRISPR/Cas9 technology was conducted for the SS18-SSX1^(+)-HEK293T cell line.Methods Lentivirus-CRISPR v2-SS18-gRNA-SSX1-gRNA-EGFP lentiviral vectors were constructed by designing single guide RNAs(sgRNAs)targeting SS18 and SSX1,respectively.The HEK293T poly The transfected cells were screened by lentiviral transfection and fluorescence flow sorting,and a SS18-SSX1^(+)-HEK293T monoclonal cell line was successfully constructed by RT-PCR,Sanger sequencing and Western Blot.Results A lentivirus-CRISPR v2-SS18-gRNA-SSX1-gRNA-EGFP lentiviral vector was successfully constructed.One SS18-SSX1 positive monoclonal cell line was successfully screened and validated in 178 HEK293T monoclonal cells by RT-PCR,Sanger sequencing and WB experiments.Conclusion First successful construction of SS18-SSX1 fusion gene positive HEK293T cell line using CRISPR/Cas9 technology.

关 键 词：CRISPR/Cas9 滑膜肉瘤 SS18-SSX1 HEK293T细胞 

分 类 号：R738[医药卫生—肿瘤]