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

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Systems Microbiology and Biomanufacturing 2023年第4期3卷 659-668页

作者： Xuefeng Ding Yun Chen Hao Wu Zhaoqi Yang Yanfei Cai Yunfeng Dai Qinghe Xu Jian Jin Huazhong Li School of Biotechnology Jiangnan UniversityWuxiPeople’s Republic of China Department of Drug Design and Molecular Pharmacology School of Life Sciences and Health EngineeringJiangnan UniversityWuxiPeople’s Republic of China

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.

关键词： CHO site-specific integration Stable expression CRISPR–Cas9 HSA

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Targeted expression of vascular endothelial growth factor 165 in the hrDNA locus mediated by hrDNA targeting vector

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Chinese Medical Journal 2007年第5期120卷 427-430页

作者： LIANG Zong-min LUO Hong YANG Yi-feng LIU Xiong-hao XIE Li XUE Zhi-gang HU Dong-xu LIANG De-sheng XIA Kun XIA Jia-hui Department of Cardiovascular Surgery Second Xiangya Hospital of Central South University Changsha 410011China National Laboratory of Medical Genetics of China Changsha 410011China Department of Respiratory Medicine Second Xiangya Hospital of Central South University Changsha 410011China

Vascular endothelial growth factor （VEGF） is an angiogenic regulator that stimulates endothelial cell migration, proliferation, and angiogenesis. VEGF gene therapy is a new promising approach to induce therapeutic angiogenesis for the treatment of ischemic myocardial and limb diseases. Recently, clinical studies have demonstrated successful outcomes using plasmids, retroviruses and adenoviruses.l-3 But the safety of those vectors is poor, which has become a serious concern. Besides immunogenicity caused by viral vectors, a further problem is that viral vectors show a preference to integrate into the transcription or control region of active genes, which may induce tumors and other disorders.

关键词： human-derived vector site-specific integration targeting expression vascular endothelial growth factor 165

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