摘要
近年来,中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞工程改造主要通过敲入或敲除基因来改变细胞某个单一功能,而敲入和敲除的基因往往无法在单次实验操作中同时发挥相应的功能,限制了多基因同步改造的应用。该研究选取细胞凋亡通路中抗凋亡蛋白即B淋巴细胞瘤-2(B-cell lymphoma-2, Bcl-2)基因为敲入基因、蛋白岩藻糖基化通路中岩藻糖合成关键酶即岩藻糖转移酶8(fucosyltransferase 8, FUT8)基因为敲除基因作为模型,利用CRISPR/Cas9技术建立定点同步敲入敲除基因编辑策略。利用该策略获得的单克隆细胞株Bcl-2蛋白过表达且FUT8蛋白酶功能缺失。经传代培养发现,由建立的定点同步敲入敲除基因编辑策略获得的细胞株在60 d内所编辑的基因表达稳定。相较于原野生型细胞,该细胞株表现出对血清剥夺的耐受度更高以及对死亡的抵抗能力更强。由此说明基于定点同步敲入敲除基因编辑策略具备一定可行性,可用于重组蛋白生产的CHO工程细胞株的构建。
In recent years,CHO(Chinese hamster ovary)cell engineering transformation mainly changes a single function of cells by knock-in or knock-out genes,but the knock-in and knock-out genes often cannot simultaneously play the corresponding functions in a single experimental operation,which limits the application of multigene synchronous transformation.In this study,Bcl-2,the anti-apoptotic gene in the apoptosis pathway,and FUT8,the key enzyme gene of fucose synthesis in the protein fucosylation pathway,were selected as the knock-in and knock-out genes.CRISPR/Cas9 technology was used to establish the site-specific synchronous knock-in and knock-out gene editing strategy.The monoclonal cell line obtained by using this strategy overexpressed Bcl-2 protein and lost FUT8 protease function.After subculture,it was found that the gene expressions of the cell lines obtained by the established site-specific synchronous knock-in and knock-out gene editing strategy were stable within 60 d.Compared with the original wild-type cells,the cell line demonstrated higher tolerance to serum deprivation and stronger resistance to death.This reveals that the gene editing strategy based on site-specific synchronous knock-in and knock-out is feasible and can be used to construct CHO engineering cell lines for recombinant protein production.
作者
程静雯
曹磊
张艳敏
叶倩
陈敏
谭文松
赵亮
CHENG Jing-wen;CAO Lei;ZHANG Yan-min;YE Qian;CHEN Min;TAN Wen-song;ZHAO Liang(State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237;Shanghai Bioengine Sci-Tech Co.,Ltd.,Shanghai 201203)
出处
《生物技术通报》
CAS
CSCD
北大核心
2023年第2期283-291,共9页
Biotechnology Bulletin
