Utilizing FACS-based Screening and UCOE Combined Strategy Accelerates
Clonal Selection and Improves Recombinant Protein Productivity in
Chinese Hamster Ovary Cells
Abstract
Utilizing reporter genes and FACS-based screening is a straightforward
and fast approach to accelerate the identification of high producer cell
lines. The genetic regulatory elements such as ubiquitous
chromatin-opening element (UCOE) could reduce the negative random
insertional effect of the expression cassette and boost the gene of
interest transcription level. Here, we used the combined strategy of
FACS-based screening by green fluorescence intensity and UCOE to
accelerate the clonal selection and enhance recombinant Darbepoetin alfa
(DPO) productivity in Chinese Hamster Ovary (CHO) cells. In this way,
two expression cassettes, pOptiVECTM and UCOE-containing plasmid,
CET1019HD, which entailed codon optimized Darbepoetin
alfa-LoxP-IRES-EGFP-LoxP-IRES-DHFR fragment, were designed. To achieve
stable cell line, the cassettes were linearized and transfected to the
CHO DG44 cells. After achieving stably transfected clones, EGFP was used
as a selection marker in FCAS to enrich the cells with the brightest
green fluorescent. In the Following, the DPO and EGFP expressions level
were assessed through qRT-PCR, FCM, western blotting, and ELISA.
Expression analysis revealed that all UCOE-containing cell pools
indicated higher DPO yield compared to non-UCOE populations. Indeed,
FACS sorting and enrichment of the UCOE-entailing cells leads to
obtaining a clone with more than 8-fold productivity. Besides, isolating
high-producing cells through FACS with a simple gate resulted in a
1.5-fold improvement of target protein concentration compared to the
unsorted cells. According to the results, we suggest the EGFP-FACS-based
screening for sorting high-producer recombinant cell lines in a reduced
time and UCOE integrated strategy to enhance protein production
dramatically.