Page 4 - K. Development and regeneration

[K. Development and regeneration-2]

Next generation vector platform for easy replacement of GOI

using RMCE-mediated site specific integration in CHO Cells

Jaewon Kim¹, Joontae Park¹˙*

¹Division of Life Sciences, Incheon National University, Incheon KS006, Korea

Chinese hamster ovary (CHO) cells are mammalian hosts commonly used in the production of biotherapeutic agents.
Traditional CHO cell line development is based on random integration of transgenes into the genome, providing
the unpredictable expression. Thus, improved approaches to establish clones that exhibit predictable levels of

expression and desirable amounts have been proposed as potentially effective strategies in the biopharmaceutical

industry. In this study, we developed a recombinase mediated cassette exchange (RMCE) system using Cre/Lox. Cells
with a landing pad (LP; containing mCherry flanked by Lox5171 and LoxP sequences), which serve as a prerequisite
for RMCE, were established by incorporating LP into predefined hotspots using CRISPR / Cas9 mediated

homologous recombination. RMCE exchanged LP with a targeting vector (containing GFP flanked by Lox5171 and
LoxP) as evidenced by the appearance of cells expressing GFP but losing mCherry. Moreover, optimal conditions for

RMCE were established by adjusting the ratio between the targeting vector and the Cre plasmid to 4:1. Furthermore,
FACS sorting enriches up to 80% GFP positive cells in which RMCE has been successfully generated, providing the

possibility of RMCE being used for commercial cell line development time.