POLQ基因敲除293T细胞系的构建及其在外源基因定点整合中的应用  

Construction of POLQ Gene Knockout 293T Cell Line and Its Application for Target Integration of Exogenous Gene

作　　者：王瑶 姜志洋 蔡军雨 樊榕榕 贾姗姗 冯健男 石艳春[1] 王晶 郑源强[1] WANG Yao;JIANG Zhiyang;CAI Junyu;FAN Rongrong;JIA Shanshan;FENG Jiannan;SHI Yanchun;WANG Jing;ZHENG Yuanqiang(Inner Mongolia Key Laboratory of Molecular Biology,Inner Mongolia Medical University,Hohhot 010058,China;National Key Laboratory of National Security Special Drugs,Beijing Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]内蒙古医科大学内蒙古自治区分子生物学重点实验室,呼和浩特010058 [2]军事科学院军事医学研究院毒物药物研究所国家安全特需药品全国重点实验室,北京100850

出　　处：《中国生物工程杂志》2024年第5期40-55,共16页China Biotechnology

基　　金：科技部国家重点研发计划(2023YFC2605002);北京市自然科学基金(7222262);内蒙古自治区自然科学基金(2022MS08011)资助项目。

摘　　要：目的:利用CRISPR/Cas9技术构建POLQ基因敲除293T细胞系,并探讨该敲除细胞系在外源基因定点整合方面的应用。方法:构建靶向POLQ基因的pX330-sgRNA打靶载体,通过T7EI酶切验证,选取打靶效率最高的载体,转染293T细胞;利用流式细胞分选术、稳定单细胞克隆培养、TA克隆测序及蛋白质印迹等方法验证POLQ敲除情况;利用靶向AAVS1和FBL位点的含GFP/mCherry报告基因同源打靶载体,评估敲除的293T POLQ-/-细胞系在外源基因定点整合效率的变化,并通过测序方式验证供体报告载体是否定点整合到基因组中;最后分析POLQ敲除对293T细胞增殖和存活率的影响。结果:成功在293T细胞中稳定敲除POLQ基因;通过报告基因实验验证,敲除细胞系中外源基因定点整合效率平均提高2~7倍,且敲除POLQ基因对293T细胞系增殖和存活率没有明显影响。结论:成功利用CRISPR/Cas9技术构建具有高效定点整合能力的POLQ基因稳定敲除293T细胞系,为进一步利用该细胞系高效制备稳定整合外源基因(包括抗体基因)工程化细胞系奠定基础。Objective:To construct POLQ gene knockout lines in 293T cells using CRISPR/Cas9 technology and to investigate its application for targeted integration of foreign genes.Methods:By constructing pX330-sgRNA targeting vector targeting POLQ gene,then after T7 endonuclease I verification,the vector with the highest targeting efficiency was selected to transfect 293T cells.Deletion of POLQ gene was confirmed by flow cytometric sorting,stable single cell clone culture,TA clone sequencing and Western blotting analysis.The GFP/mCherry homologous reporter vector targeting the AAVS1 and FBL sites was used to evaluate the efficiency of targeted integration of the exogenous gene in the knockout 293T POLQ-/-cell line,and the targeted integration of the donor reporter vector into the genome was verified by sequencing.Finally,the effects of POLQ gene knockout 293T cells on cell proliferation and survival rate were evaluated.Results:The POLQ gene was stably knocked out in 293T cells.Validation by reporter gene assays demonstrated that the efficiency of target integration of exogenous genes in the knockout cell line increased by an average of 2-fold to 7-fold.Furthermore,knocking out the POLQ gene had no significant effect on the proliferation and survival rate of 293T cells.Conclusion:Using CRISPR/Cas9 technology,a stable POLQ gene knockout cell line was successfully established in 293T cells,and the efficient targeted integration of exogenous genes was achieved.These findings provide a basis for using the cell line to generate stable integration of foreign genes,including antibody genes,facilitating the advancement of engineered cellular systems.

关 键 词：CRISPR/Cas9 POLQ 293T细胞 基因敲除 定点整合 

分 类 号：Q812[生物学—生物工程]