Cre-mediated cellular modification for establishing producer CHO cells of recombinant scFv-Fc

Abstract

The targeted integration of transgenes into a pre- characterized genomic locus enables predictable pro- tein expression to occur, which reduces the need for the screening of transfected clones. We previously developed an accumulative site-specific gene integra- tion system (AGIS), which enabled the repeated inte- gration of multiple transgenes into a pre-determined locus of the cell genome [1,2]. We achieved the repeated integration of recombinant scFv-Fc gene into the genome of Chinese hamster ovary (CHO) cells, a common animal host cell for the production of recom- binant biopharmaceutical proteins. Productivity was shown to correspond to the copy number of the expres- sion cassette [3]. Cell screening after gene transfection was the most time-consuming process in AGIS, but we hypothesized that the use of fluorescent selection mar- kers instead of drug resistant genes would facilitate the cell establishment process because Cre-mediated inte- gration is completed within 48 h post-transfection. Therefore, the present study used marker genes encod- ing fluorescent proteins to speed up the establishment of producer CHO cells using AGIS.