• 中国精品科技期刊
  • 中国高校百佳科技期刊
  • 中国中文核心期刊
  • 中国科学引文数据库核心期刊
Advanced Search
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
Citation: 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
shu

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

More Information
  • Received Date: February 07, 2021
  • Revised Date: April 18, 2021
    Graphical 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.
    • FullText(HTML)
    • References (18)
  • [1]
    . Biotechnol Adv，2015，33：1878-1896.
    [2]
    Peng L，Yu X，Cai YF，et al. Research advances of recombinant coagulation factor VII expression and synthesizing mechanism［J］.J China Pharm Univ（中国药科大学学报），2015，46（5）：623-628.
    [3]
    Hamaker NK，Lee KH. Site-specific integration ushers in a new era of precise CHO cell line engineering［J］. Curr Opin Chem Eng，2018，22：152-160.
    [4]
    Lee JS，Grav LM，Pedersen LE，et al. Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment［J］. Biotechnol Bioeng，2016，113（11）：2518-2523.
    [5]
    Cheng JK，Lewis AM，Kim DS，et al. Identifying and retargeting transcriptional hot spots in the human genome［J］. Biotechnol J，2016，11（8）：1100-1109.
    [6]
    Yang L. Study on site-specific integration and expression of human serum albumin in CHO-K1 cell genome［D］.Jiangnan University，2020.
    [7]
    Zhou ST. Study of site-specific integration of therapeutic protein genes into CHO genome and stable expression［D］.Jiangnan University，2019.
    [8]
    Plesingerova H，Janovska P，Mishra A，et al. Expression of COBLL1 encoding novel ROR1 binding partner is robust predictor of survival in chronic lymphocytic leukemia［J］. Haematologica，2018，103（2）：313-324.
    [9]
    Chen ZF，Yu HJ，Shi X，et al. Functional screening of candidate causal genes for insulin resistance in human preadipocytes and adipocytes［J］. Circ Res，2020，126（3）：330-346.
    [10]
    Sharma V，Sharma I，Sethi I，et al. Replication of newly identified type 2 diabetes susceptible loci in Northwest Indian population［J］. Diabetes Res Clin Pract，2017，126：160-163.
    [11]
    Park JW，Jang HJ，Shin S，et al. Molecular analysis of alternative transcripts of the equine cordon-bleu WH2 repeat protein-like 1 （COBLL1） gene［J］. Asian-Australas J Anim Sci，2015，28（6）：870-875.
    [12]
    Chauhan D，Bartok E，Gaidt MM，et al. BAX/BAK-induced apoptosis results in caspase-8-dependent IL-1beta maturation in macrophages［J］. Cell Rep，2018，25（9）：2354-2368.
    [13]
    O''Neill KL，Huang K，Zhang JJ，et al. Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane［J］. Genes Dev，2016，30（8）：973-988.
    [14]
    Zhao SQ，Zhang Y，Tian H，et al. Expression and bioactivity of recombinant 3DHSA-Nartograstim fusion protein in Pichia pastoris［J］. J China Pharm Univ（中国药科大学学报），2013，44（6）：577-582.
    [15]
    Bailey LA，Hatton D，Field R，et al. Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture［J］. Biotechnol Bioeng，2012，109（8）：2093-2103.
    [16]
    Shin SW，Lee JS. Optimized CRISPR/Cas9 strategy for homology-directed multiple targeted integration of transgenes in CHO cells［J］. Biotechnol Bioeng，2020，117（6）：1895-1903.
    [17]
    Zhou ST，Ding XF，Yang L，et al. Discovery of a stable expression hot spot in the genome of Chinese hamster ovary cells using lentivirus-based random integration［J］. Biotechnol Biotec Eq，2019，33（1）：605-612.
    [18]
    Gaidukov L，Wroblewska L，Teague B，et al. A multi-landing pad DNA integration platform for mammalian cell engineering［J］. Nucleic Acids Res，2018，46（8）：4072-4086.
    • Related Articles

  • Related Articles

    [1]WANG Chi, TIAN Hong, GAO Xiangdong, YAO Wenbing. Influence of ubiquitous chromatin opening element on the antibody expression in CHO cells[J]. Journal of China Pharmaceutical University, 2017, 48(1): 96-101. DOI: 10.11665/j.issn.1000-5048.20170115
    [2]BI Lei, GAO Jing, JIANG Yucui, CHEN Yingna, CHEN Weiping. Effect of Ginseng, Ginseng polysaccharides and Ginsenoside on the human lung cancer cells A549 in co-culture system of TAMs with A549 cells[J]. Journal of China Pharmaceutical University, 2016, 47(6): 744-748. DOI: 10.11665/j.issn.1000-5048.20160619
    [3]LIU Su, TIAN Hong, WANG Chi, YAO Wenbing. Weakening resistance marker for establishing a process of screening high-producing CHO cell lines[J]. Journal of China Pharmaceutical University, 2015, 46(5): 617-622. DOI: 10.11665/j.issn.1000-5048.20150517
    [4]WANG Xinning, XU Bin, ZHOU Jinpei, ZHANG Huibin. Advances of anti-diabetic drugs based on new targets[J]. Journal of China Pharmaceutical University, 2015, 46(2): 141-152. DOI: 10.11665/j.issn.1000-5048.20150202
    [5]GAO Yuan, ZHAO Yue, YAO Jing, WANG Yu, GUO Qinglong, WU Yulin. Wogonin suppresses human alveolar adenocarcinoma cell A549 invasion and metastasis induced by IL-6[J]. Journal of China Pharmaceutical University, 2014, 45(4): 456-461. DOI: 10.11665/j.issn.1000-5048.20140413
    [6]MENG Yu-han, LIU Su-li, ZHANG Hui-yong, CAO Rong-yue, FAN Xiao-xu, FAN Hao, LIU Jing-jing. Construction,expression and activity studies of recombinant GFP-VEGF6a fusion polypeptide[J]. Journal of China Pharmaceutical University, 2011, 42(1): 83-87.
    [7]SHI Wei, SUN Li, LIN Sen-sen, YUAN Sheng-tao, ZHANG Lu-yong. Construction of eukaryotic expression plasmid for human TF full-length，TF truncated and characterization of their biological function[J]. Journal of China Pharmaceutical University, 2009, 40(5): 465-470.
    [8]GUO Zhong, ZHAO Jin, MA Jian-xiu, FU Si-wu. Effects of toxin A from Clostridium difficile on SMMC-7721 cells proliferation and apoptosis[J]. Journal of China Pharmaceutical University, 2009, 40(3): 250-253.
    [10]Effect of Lomerizine on Multidrug Resistance in Human Leukemic Cell Line K562/A02[J]. Journal of China Pharmaceutical University, 2003, (4): 70-74.

  • Cited by

    Periodical cited type(2)

    1. 杨振苹，邢体坤，宋路萍，刘伟，李艳芳，张静静. TaqMan荧光定量PCR法检测CHO细胞中外源基因拷贝数的方法建立. 中国当代医药. 2023(02): 15-19 .
    2. 瞿丽丽，丁学峰，蔡燕飞，鲁晨，李华钟，金坚，陈蕴. CHO细胞基因组NW-003614092.1内稳定表达位点的发现. 中国生物工程杂志. 2022(06): 12-19 .

    Other cited types(3)

Catalog

    Get Citation
    PDF
    XML
    Article views (216) PDF downloads (918) Cited by(5)
    Turn off MathJax
    Article Contents
    Abstract
    References
    Related Articles

    Copyright © Journal of China Pharmaceutical University苏ICP备12050101号-2

    Address：24 Tongjia Lane, Nanjing City, Jiangsu Province （210009）Tel: 025-83271566E-mail: xuebao@cpu.edu.cn

    Supported by: Beijing Renhe Information Technology Co., Ltd.  Baidu Analytics

    Total visits：297451

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return