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中国生物工程杂志

China Biotechnology
China Biotechnology  2022, Vol. 42 Issue (3): 72-81    DOI: 10.13523/j.cb.2107043
    
Research Progress on Stable Hot Spot Sites in the Genome of CHO Cells
LIN Jian-fen,LUO Shun**()
Jiangxi University of Chinese Medicine, Nanchang 330004, China
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Abstract  

In recent years, therapeutic recombinant protein drugs have become a research hot topic in the field of biopharmaceuticals research. Chinese hamster ovary cell lines are commonly used for recombinant protein expression in industrial production. The traditional expression of CHO cell lines is mostly based on random integration. However, random integration may result in target genes targeted into heterochromatin regions or unstable chromatin regions, causing unstable expression in CHO cell lines, and in order to obtain the ideal expression cell line researchers have to do multiple rounds of screening. The latest research proves that GOI integrates specifically in the prediction or specific genomic sites of CHO cells, which can maintain long-term consistency and stability in the expression of recombinant CHO cells. The integration sites for efficient and stable transcription in the genome of CHO cells are called hot spots. This review describes the latest research progress of stable hot spot sites in the CHO cell genome, summarizes the research methods of these sites, and concludes how to locate the GOI at predicted gene locus efficiently, to achieve high-level and stable expression recombinant protein, and to help researchers discover new or effective hot spot sites and then construct stable expression CHO cell lines.



Key wordsCHO      Hot spot      Site-specific integration      Cell line development     
Received: 19 July 2021      Published: 07 April 2022
ZTFLH:  Q819  
Corresponding Authors: Shun LUO     E-mail: shun.luo@tobiopharm.com
Cite this article:

LIN Jian-fen, LUO Shun. Research Progress on Stable Hot Spot Sites in the Genome of CHO Cells. China Biotechnology, 2022, 42(3): 72-81.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2107043     OR     https://manu60.magtech.com.cn/biotech/Y2022/V42/I3/72

Fig.1 Construction of recombinant CHO cell line (a) Raditional CLD random integration (b) Site-specific integration method
Fig.2 Graph showing the number of publications on different subtopics related to the theme of site specific integration in CHO Data from pubmed (May 2021) by serching “site specific integration, target integration and hot spot”, in combination with the keyword “CHO”
整合位点 细胞类型 目的基因 参考文献
Rosa26 CHO-K1 mAb-JUG444 (Pfizer) [25]
HEK293T GFP (Green fluorescent protein)gene [27]
Hprt CHO-K1 Red fluorescence protein gene [28-29]
CHO-K1 pFW (5.3 kb) [30]
CHO-K1 scFv-Fc antibody gene [31]
Fer1L4 CHO-S EGFP (Enhanced Green Fluorescent Protein) [32]
CHOK1SV IgG2 mAb Myo (Pfizer) gene [33]
C1GALT1C1(COSMC) CHO-S mCherry [34]
HEK293 EGFP [35]
CHO-S GFP gene [36]
NW_006880285.1 HEK-293T Zsgreen1 reporter gene [10]
C12orf35 CHO-K1 mCherry and anti-PD1 mAb gene [24]
Putative Sites T2 and T9 CHO-S Rituximab gene [37-38]
FRT site CHO-DHFR tissue plasminogen activator (tPA) gene [39]
FUT8 CHO-K1 mAb gene [40]
Hipp11 CHO-S GFP gene [27]
Table 1 Confirmed hot spot loci in the CHO genome
Fig.3 The principles of GOI site-specific integration (a) Nuclease-mediated (b) Recombinase-mediated
方法 优势 缺点 参考文献
重组酶介导的位点
特异性整合
缩短细胞系开发的时间
保证外源基因的表达水平和稳定表达
有多个外源基因重组位点
不使用多组学数据确定hot spot位点
可构建重复靶向的主细胞系平台
基因框架在预定的靶点可以有效地表达
靶向整合GOI前RTS需要提前构建
landing pads的随机整合
重组效率低
目标重组细胞系的获得需要多轮筛选
[14,16,23,34,41]
核酸酶介导的位点
特异性整合
外源基因的高保真整合
可介导单个基因组的整合进入多个宿主细胞基因组位点
外源基因整合入宿主基因组时具有方向性
多个外源基因组片段可同时靶向供体DNA
同源重组效率低
可能会出现脱靶现象
双链断裂处基因重组是可能会出现异常
[11, 37-38,41,46, 49]
Table 2 Comparison of GOI site-specific integration methods
Fig.4 CRISPR/Cas9 and RMCE mediate GOI site-specific integration
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