	US 11,692,204 B2
	Use of genomic NW_006880285.1 in CHO cell for stably expressing a protein
Jian Jin, Wuxi (CN); Yun Chen, Wuxi (CN); Huazhong Li, Wuxi (CN); Songtao Zhou, Wuxi (CN); Zuoying Duan, Wuxi (CN); and Xiaohai Gong, Wuxi (CN)
Assigned to JIANGNAN UNIVERSITY, Wuxi (CN)
Appl. No. 16/644,953
Filed by Jiangnan University, Wuxi (CN)
PCT Filed Dec. 3, 2018, PCT No. PCT/CN2018/118887 § 371(c)(1), (2) Date Mar. 5, 2020, PCT Pub. No. WO2020/087640, PCT Pub. Date May 7, 2020.
Claims priority of application No. 201811274680.0 (CN), filed on Oct. 30, 2018.
Prior Publication US 2021/0222202 A1, Jul. 22, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/90 (2006.01); C12N 15/66 (2006.01)

CPC C12N 15/907 (2013.01) [C12N 15/66 (2013.01); C12N 2310/20 (2017.05); C12N 2510/02 (2013.01); C12N 2800/80 (2013.01)]

13 Claims

1. A method for stably expressing a protein at a predetermined site in a Chinese hamster ovary (CHO) cell genome, wherein the predetermined site in the CHO cell genome for stably expressing the protein is positioned at a base of No. 1235357 in a CHO cell gene NW_006880285.1; the method comprises:

identifying a nucleotide sequence of SEQ ID NO: 15 in a base range of No. 1235284-1235429 in the CHO cell gene NW_006880285.1, the base range having the nucleotide sequence of SEQ ID NO: 12, around the predetermined site by CRISPR/Cas9 technology as a target sequence,

transforming the CHO cell with a recombinant donor carrier containing the target sequence to obtain a recombinant CHO cell;

culturing the recombinant CHO cell and collecting the supernatant to detect the expression level, and adapting an adherent recombinant CHO cell to a suspension culture; and

culturing the adapted recombinant CHO cell.