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Discovery of Stable Expression Sites in CHO Genome NW-003614092.1 |
QU Li-li1,DING Xue-feng2,CAI Yan-fei1,LU Chen1,LI Hua-zhong2,JIN Jian1,CHEN Yun1,**() |
1. School of Life Science and Health Engineering, Jiangnan University, Wuxi 214122, China 2. School of Biotechnology, Jiangnan University, Wuxi 214122, China |
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Abstract Objective: The purpose is to provide a stable expression site with clear information for constructing a Chinese hamster ovary cell (CHO) line stably expressing recombinant protein by site-specific integration and shortening the research and development timeline. Methods: The CHO-K1-1d2 cell line with potential stable expression site randomly integrated with Zsgreen1 gene by lentivirus was continuously subcultured to verify the stability of expression; the integration site of lentiviral vector was analyzed by chromosome walking, and the editability of the site was verified by CRISPR/Cas9 technology. Results: 100% of CHO-K1-1d2 cells could emit green fluorescence and the fluorescence intensity was stable in the process of continuous adherent culture for 20 generations and suspension culture for 50 generations, indicating that Zsgreen1 protein could be stably expressed. The sequence results in chromosome walking analysis showed that the antiviral vectors were integrated between bases 1 159 463 and 1 159 467 of the CHO cell genome NW-003614092.1. The sequence results after co-transfection of sgRNA and Cas9 plasmid in CHO-K1 cells showed that this site can be edited by CRISPR/Cas9 technology. Conclusion: There is a stable expression site in CHO cell genome NW-003614092.1, which has clear information and can be edited by CRISPR/Cas9 technology.
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Received: 21 February 2022
Published: 07 July 2022
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Corresponding Authors:
Yun CHEN
E-mail: chenyun72@126.com
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