CHO细胞基因组NW-003614092.1内稳定表达位点的发现  

Discovery of Stable Expression Sites in CHO Genome NW-003614092.1

作　　者：瞿丽丽 丁学峰 蔡燕飞 鲁晨 李华钟[2] 金坚 陈蕴 QU Li-li;DING Xue-feng;CAI Yan-fei;LU Chen;LI Hua-zhong;JIN Jian;CHEN Yun(School of Life Science and Health Engineering,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生命科学与健康工程学院,无锡214122 [2]江南大学生物工程学院,无锡214122

出　　处：《中国生物工程杂志》2022年第6期12-19,共8页China Biotechnology

基　　金：国家高技术研究发展计划(2015AA020802)资助项目。

摘　　要：目的:获得中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)内稳定表达位点信息,为构建重组蛋白CHO稳定表达株、缩短研发时间线提供信息明确的稳定位点。方法:对慢病毒随机整合Zsgreen1基因的具有潜在稳定表达位点的CHO-K1-1d2细胞株进行连续传代培养,验证表达稳定性;通过染色体步移分析慢病毒载体整合位点,并利用CRISPR/Cas9技术验证位点的可编辑性。结果:CHO-K1-1d2细胞在连续贴壁培养20代、悬浮培养50代过程中,能够100%发绿色荧光,且荧光强度稳定,能够稳定表达Zsgreen1蛋白;染色体步移分析测序结果表明,CHO-K1-1d2细胞中慢病毒载体整合于CHO细胞基因组NW-003614092.1上第1159463与1159467碱基间;共转染sgRNA与Cas9质粒后,测序结果表明,该位点可被CRISPR/Cas9技术编辑。结论:CHO细胞基因组NW-003614092.1内存在一个信息明确的、能够被CRISPR/Cas9技术编辑的稳定表达位点。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-K1-1 d2 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-K1-1 d2 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 1159463 and 1159467 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.

关 键 词：CHO 稳定位点 染色体步移 CRISPR/Cas9 

分 类 号：Q813[生物学—生物工程]