Premium
An Efficient Exogenous Gene Insertion Site in CHO Cells with High Transcription Level to Enhance AID‐Induced Mutation
Author(s) -
Fan Yingjun,
Jiang Wei,
Ran Fanlei,
Luo Ruiqi,
An Lili,
Hang Haiying
Publication year - 2020
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201900313
Subject(s) - chinese hamster ovary cell , somatic hypermutation , cytidine deaminase , biology , activation induced (cytidine) deaminase , gene , microbiology and biotechnology , transcription (linguistics) , clone (java method) , mutation , genetics , cell culture , antibody , b cell , linguistics , philosophy
Antibodies have been extensively used for the purpose of scientific research, clinical diagnosis, and therapy. Combination of in vitro somatic hypermutation and mammalian cell surface display has been an efficient technology for antibody or other proteins optimization, in which the efficiency of activation‐induced cytidine deaminase (AID) mutations in genes is one of the most important key factors. Gene transcriptional level has been found to be positively proportional to AID‐induced mutation frequency. Thus, construction of the cell clone bearing a gene of interest (GOI) with high transcription level can increase AID‐induced mutations. In this study, a retargetable gene cassette is inserted onto predetermined chromosome site ( ywhae gene site) which is among the genes with the highest as well as stable transcription, and is found that one subsite is suitable to be retargeted for efficient protein display in Chinese hamster ovary (CHO) cells. The resultant cell clone (T31) has higher and more stable transcription/expression than CHO‐puro clone which was previously established through the strategy of random insertion followed by a high‐throughput selection. It also possesses a significantly higher mutation frequency to GOI than CHO‐puro cells; thus, it is a better clone for the in vitro improvement of antibody affinity, and probably other properties.