An Efficient Exogenous Gene Insertion Site in CHO Cells with High Transcription Level to Enhance AID-Induced Mutation.
CRISPR/Cas9
Chinese hamster ovary cells
activation-induced cytidine deaminase
high protein expression/display
recombinase-mediated cassette exchange
Journal
Biotechnology journal
ISSN: 1860-7314
Titre abrégé: Biotechnol J
Pays: Germany
ID NLM: 101265833
Informations de publication
Date de publication:
May 2020
May 2020
Historique:
received:
22
07
2019
revised:
18
12
2019
pubmed:
25
1
2020
medline:
22
12
2020
entrez:
25
1
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 31975519
doi: 10.1002/biot.201900313
doi:
Substances chimiques
Green Fluorescent Proteins
147336-22-9
AICDA (activation-induced cytidine deaminase)
EC 3.5.4.-
Cytidine Deaminase
EC 3.5.4.5
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e1900313Subventions
Organisme : Beijing Science and Technology Project
ID : Z181100003818023
Organisme : National Natural Science Foundation of China
ID : 31500753
Organisme : National Natural Science Foundation of China
ID : 31870923
Informations de copyright
© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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