Site-specific integration and stable expression of exogenous protein at a novel site on CHO cell chromosome

HU Wanwan; DING Xuefeng; CAI Yanfei; CHEN Yun; DUAN Zuoying; JIN Jian; LI Huazhong

doi:10.11665/j.issn.1000-5048.20210412

Journal of China Pharmaceutical University > 2021 > 52(4): 487-495. > DOI: 10.11665/j.issn.1000-5048.20210412

HU Wanwan, DING Xuefeng, CAI Yanfei, CHEN Yun, DUAN Zuoying, JIN Jian, LI Huazhong. Site-specific integration and stable expression of exogenous protein at a novel site on CHO cell chromosome[J]. Journal of China Pharmaceutical University, 2021, 52(4): 487-495. DOI: 10.11665/j.issn.1000-5048.20210412

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Site-specific integration and stable expression of exogenous protein at a novel site on CHO cell chromosome

1.
School of Biotechnology, Jiangnan University, Wuxi 214122
2.
School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China

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Received Date: February 07, 2021
Revised Date: April 18, 2021

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Abstract

Abstract

Finding stable expression sites on the chromosomes of Chinese hamster ovary (CHO) cells is an effective method to solve the problem of unstable expression of CHO cells in long-term culture. Our group used lentiviral transfection to integrate the tracer gene (Zsgreen1) into the chromosome of CHO cells and found multiple potential stable expression sites. This study verified the ability of one of the sites located in the 148052-148157 bp region on chromosome NW_003614241.1 to stably express exogenous proteins.The expression of Zsgreen1 gene was first observed, and CRISPR/Cas9 technology was then used to integrate the enhanced green fluorescent protein (EGFP) gene into this site. Three strains of EGFP gene integrated cells were obtained. After 60 generations of suspension culture, the fluorescence intensity of the cells had no significant changes, which proved that this site can stably express the EGFP gene. The same method was used to construct recombinant CHO cell lines expressing the human serum albumin (HSA) gene, and was verified by Western blot that this site could express and secrete HSA. It shows that the above-mentioned sites can be integrated and can stably express exogenous proteins.
- CRISPR/Cas9,
- site-specific integration,
- stable expression,
- a novel site,
- Chinese hamster ovary (CHO) cells

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