Discovery of Stable Expression Sites in CHO Genome NW-003614092.1

doi:10.13523/j.cb.2202035

China Biotechnology

2022, Vol. 42

Issue (6): 12-19 DOI: 10.13523/j.cb.2202035

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 Yun^1,^**(

)

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.

Key words： CHO Stable sites Genome walking CRISPR/Cas9

Received: 21 February 2022 Published: 07 July 2022

ZTFLH:

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Corresponding Authors: Yun CHEN E-mail: chenyun72@126.com

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	Li-li QU
	Xue-feng DING
	Yan-fei CAI
	Chen LU
	Hua-zhong LI
	Jian JIN
	Yun CHEN

Cite this article:

QU Li-li,DING Xue-feng,CAI Yan-fei,LU Chen,LI Hua-zhong,JIN Jian,CHEN Yun. Discovery of Stable Expression Sites in CHO Genome NW-003614092.1. China Biotechnology, 2022, 42(6): 12-19.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2202035 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I6/12

Table 1 Primer sequences for genome walking

Table 2 Primer sequences for site verification

Fig.1 Schematic diagram of integration site verification

Fig.2 Expression of Zsgreen1 protein in CHO-K1-1d2 cells cultured in adherent for 20 passages(a) Growth curve of CHO cells (b) Inverted fluorescence microscope to observe the expression of Zsgreen1 protein (c) ImageJ software analysis results (d), (e) Quantitative detection of Zsgreen1 protein expression by flow cytometry

Fig.3 Expression of Zsgreen1 protein in CHO-K1-1d2 cells cultured in suspension for 50 passages(a) Inverted fluorescence microscope to observe the expression of Zsgreen1 protein (b) ImageJ software analysis results (c), (d) Quantitative detection of Zsgreen1 protein expression by flow cytometry

Fig.4 Results of agarose gel electrophoresis of secondary PCR productsM: DL15 000 DNA marker; Lane 1: Dra I digestion library; Lane 2: Ssp I digestion library; Lane 3: Hpa I digestion library

Table 3 Sequencing results of secondary PCR products

Fig.5 The results of agarose gel electrophoresis of PCR products for verification of integration sitesM: DL5 000 DNA marker; Lane 1: PCR product with SP1 and ZsP1 primers; Lane 2: PCR product with SP2 and ZsP2 primers

Table 4 Site verification sequencing results

Fig.6 Verification of site editability(a) Peak map of sgRNA expression plasmid sequencing (b) Cell pool sequencing peak map of genomic PCR products (c) Result of T7E1 enzyme digestion. M: DL5 000 marker; Lane 1: PCR product of cell pool genome; Lane 2: PCR product of cell pool digested by T7E1 enzyme


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