An Efficient Exogenous Gene Insertion Site in CHO Cells with High Transcription Level to Enhance AID‐Induced Mutation

Abstract

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.

Construction and application of a multifunctional CHO cell platform utilizing Cre/lox and Dre/rox site-specific recombination systems

“…The transcriptional activity of a gene locus is controlled by its chromatin state and genetic instability, and sites in the genome that are abnormally stable and have high transcriptional activity are called “hotspots” by researchers ( Thomas et al, 1986 ; Koduri et al, 2001 ). Many efforts have been made to identify transcriptional hotspots in host genomes and dozens of CHO hotspots have been reported, including Fer1L4, Rosa26, Ywhae, Hprt, Hipp11 and C12orf35 ( Gaidukov et al, 2018 ; Hamaker and Lee, 2018 ; Zhao et al, 2018 ; Chi et al, 2019 ; Fan et al, 2020 ; Kim et al, 2021 ). The Hipp11 ( H11 ) locus is a non-coding sequence situated between the conserved DRG1 and EIF4ENIF1 genes ( Hippenmeyer et al, 2010 ).…”