Construction of a CHO cell line with site‑specific integration to stably express exogenous proteins using the CRISPR–Cas9 technique

作     者：Xuefeng Ding Yun Chen Hao Wu Zhaoqi Yang Yanfei Cai Yunfeng Dai Qinghe Xu Jian Jin Huazhong Li 

作者机构：School of BiotechnologyJiangnan UniversityWuxiPeople’s Republic of China Department of Drug Design and Molecular PharmacologySchool of Life Sciences and Health EngineeringJiangnan UniversityWuxiPeople’s Republic of China 

出 版 物：《Systems Microbiology and Biomanufacturing》 (系统微生物学与生物制造（英文）)

年 卷 期：2023年第3卷第4期

页      面：659-668页

核心收录：

学科分类：0710[理学-生物学] 1007[医学-药学(可授医学、理学学位)] 100705[医学-微生物与生化药学] 07[理学] 071005[理学-微生物学] 10[医学] 

基　　金：School of Bioengineering Fudan University Jiangnan University, JU Novo Nordisk 

主　　题：CHO Site-specific integration Stable expression CRISPR–Cas9 HSA 

摘      要：Chinese hamster ovary(CHO)cells are widely used in biopharmaceuticals because of their high-density suspension culture,high safety,and high similarity between expressed exogenous proteins and natural ***,the level of exogenous protein expression decreases with increasing culture time;this phenomenon occurs due to the recombination of foreign genes into chromosomes through random *** present study integrated the foreign genes into a specific chromosomal site for stable expression based on CRISPR–Cas9 *** results showed that the exogenous proteins enhanced green fluorescent protein(EGFP)and human serum albumin(HSA)were successfully integrated into the vicinity of base 1969647 on chromosome NW_003613638.1 of CHO-K1 *** obtained positive monoclonal cell lines expressed all the corresponding exogenous proteins after 60 consecutive passages,and no significant differences in expression levels were *** study might provide a feasible method to construct a CHO cell line with long-term stable expression of exogenous proteins.