基于PiggyBac转座系统构建稳定表达ABEmax基因的HEK293T细胞系  

Construction of HEK293T cell line stably expressing ABEmax gene based on Piggy Bac transposition system

作　　者：张璐 安莉莎 金孝华 马旭 ZHANG Lu;AN Lisha;JIN Xiaohua;MA Xu(National Research Institute for Family Planring,Beijing,100081;National Human Genetic Resources Cen-ter,Beijing,102206)

机构地区：[1]国家卫生健康委科学技术研究所,北京100081 [2]国家人类遗传资源中心

出　　处：《中国计划生育学杂志》2022年第7期1472-1475,共4页Chinese Journal of Family Planning

基　　金：国家卫生健康委科学技术研究所中央级公益性科研院所基本科研业务费专项资金(2021GJM02)。

摘　　要：目的:使用PiggyBac(PB)转座系统在HEK293T细胞中敲入腺嘌呤碱基编辑工具ABEmax基因,构建稳定表达ABEmax基因的工具细胞系.方法:根据PB转座原理,将ABEmax基因和抗生素筛选基因以同源重组的方法插入到PB质粒内,构建pPB-ABEmax真核重组表达质粒.将重组质粒和辅助质粒共同转染至HEK293T细胞中,利用嘌呤霉素抗性筛选细胞,流式细胞仪分选单克隆细胞.单细胞扩增培养后,利用PCR鉴定ABEmax基因的插入水平,RT-qPCR鉴定ABEmax的表达水平以及利用sgRNA测试工具细胞的编辑水平.结果:成功构建pPB-ABEmax真核重组表达质粒,筛选出特异性插入ABEmax基因的HEK293T细胞系,HEK293T-ABEmax的基因编辑效率与HEK293T细胞转染Test-sgRNA与ABEmax的对照组无统计学差别(P>0.05).结论:利用PiggyBac基因编辑技术成功构建并获得了腺嘌呤碱基编辑工具ABEmax基因稳定表达细胞系.Objective: To construct a tool cell line that stably expresses the ABEmax gene based on the knocked in the adenine base editing tool ABEmax gene by HEK293 T cells PiggyBac(PB) transposition system. Methods: ABEmax gene and antibiotic screening gene were inserted into PB plasmid by homologous recombination method to construct pPB-ABEmax eukaryotic recombinant expression plasmid. The recombinant plasmids and the helper plasmids were co-transfected into HEK293 T cells, the cell lines were screened by puromycin resistance, and the monoclonal cells were sorted by flow cytometry. After expanded culture, PCR was used to identify the insertion level of the ABEmax gene, RT-qPCR to identify the expression level of ABEmax, and sgRNA transfection was used to test the editing efficiency of the tool cells. Results: The pPB-ABEmax recombinant expression plasmid was successfully constructed, the HEK293 T cell lines with specific insertion of the ABEmax gene were screened out, and the gene editing ability of the HEK293 T-ABEmax was verified. Conclusion: Several cell lines stably expressing the adenine base editing tool ABEmax gene were successfully constructed and obtained by the PiggyBac gene editing technology, and which had the same high editing activity compared with normal HEK293 T cells transfected with sgRNA and ABEmax.

关 键 词：PiggyBac转座系统 碱基编辑 ABEmax 细胞系 

分 类 号：Q78[生物学—分子生物学]