HEK293T细胞中敲除DNMT3a及其对蛋白表达的影响

【作者】 王佳贤； 丁凯； 宗会芳； 朱晨岑； 赵梦琳； 路慧丽； 朱建伟；

【机构】 上海交通大学药学院细胞工程及抗体药物教育部工程研究中心；

【摘要】 本文运用Crispr/Cas9技术在HEK293T细胞中敲除DNA甲基转移酶3a(DNMT3a)基因,并成功获得了一株完全敲除DNMT3a的细胞株HEK293T-KO。为了研究DNMT3a敲除对细胞表达重组蛋白的影响,以GFP作为模式蛋白,在HEK293T-WT和-KO细胞株分别转染表达EGFP的质粒,观察并比较转染效率和蛋白表达水平。结果显示,细胞中敲除DNMT3a基因会严重影响细胞的生长;并且与野生型细胞株相比,HEK293T-KO细胞株的转染效率显著降低;进一步采用流式细胞术检测转染48h之后GFP的荧光强度作为细胞平均GFP表达量的指标,发现敲除细胞株的平均绿色荧光强度也显著低于野生型细胞株。我们的数据说明DNMT3a对外源蛋白在HEK293T细胞中的表达具有重要作用。更多还原

【Abstract】 Crispr/Cas9 technology was used to disrupt DNA methyltransferase 3 a(DNMT3 a) gene in HEK293 T cells,and finally one cell line with DNMT3 a gene completely disrupted was obtained,which was referred to as HEK293 T-KO.To investigate the effect of DNMT3 a knockout on the recombinant protein expression,GFP was employed as the model protein and plasmids bearing EGFP encoding gene were transfected into both HEK293 T-WT and-KO cells,followed by comparing the transfection efficiency and protein productivity.And the results demonstrated that disruption of DNMT3 a gene in HEK293 T cells could influence the cell growth significantly.Compared with the wild type cell line,the transfection efficiency of HEK293 T-KO cells was much lower.As a further step,the GFP mean fluorescence intensity 48 hours post transfection was detected with flow cytometry and served as the indicator of mean GFP expression.It showed that the mean fluorescence intensity of HEK293 T-KO cells was significantly lower than that of-WT cells.Our data suggested that DNMT3 a gene played a crucial role in the exogenous protein expression.更多还原