摘要
构建敲除DNMT3B基因的细胞系,为重组蛋白药物生产使用的高效表达系统提供DNMT3B基因缺失的稳定细胞株.采用CRISPR/Cas9基因编辑技术,构建敲除DNMT3B基因的质粒,并利用脂质体转染CHO-K1细胞进行基因编辑、T7E1核酸内酶切及筛选DNMT3B基因敲除细胞,以及软琼脂法获得单克隆.结果表明,通过对获得的单细胞克隆进行测序比对,确定DNMT3B基因第4个外显子处被编辑产生移码突变.最终获得DNMT3B基因特异位点删除的CHO细胞单克隆.
Cell lines with DNMT3 B gene knockout were constructed to provide the stable productive cells of efficient expression system for recombinant protein-drug production. CRISPR/Cas9 gene editing technology was used to construct a plasmid to knocked out DNMT3 B gene, and CHO-K1 cells were transfected with liposomes for gene editing. DNMT3 B gene knockout cells were verified by T7 E1 enzyme and screened by soft AGAR method to obtain monoclones. The results showed that DNA sequencing analysis confirmed that the fourth exon of DNMT3 B gene was edited to produce frameshift mutation. Then monoclones of CHO cells which DNMT3 B gene deleted in specific locus were obtained.
作者
陈奇娜
王斌
郭庆
王静
黄伟
王霄
纪华
CHEN Qina;WANG Bin;GUO Qing;WANG Jing;HUANG Wei;WANG Xiao;JI Hua(College of Pharmacy,Dali University,Dali,Yunnan,China 671000;School of Agriculture and Life Sciences,Kunming University,Kunming,Yunnan,China 650214;Department of Oncology,920th Hospital of PLA Joint Logistic Support Force,Kunming,Yunnan,China 650032)
出处
《昆明学院学报》
2021年第6期114-119,共6页
Journal of Kunming University
基金
国家自然科学基金(31760256,32060147)
云南省地方高校联合基金(202001BA070001-217)
云南省教育厅科学研究基金资助项目(2020J0510)。
