UCOE-Mediated Transcriptional Stabilization Combined with EGFP-Based FACS Screening Enables Efficient Selection of High-Producing CHO Cells.

Position effects and transgene silencing remain major obstacles to the generation of stable high-producing CHO cell lines. Applying genetic regulatory elements, such as a ubiquitous chromatin-opening element (UCOE), can mitigate these limitations by maintaining a transcriptionally permissive chromatin environment and supporting sustained transgene expression. In parallel, EGFP-based fluorescence-activated cell sorting (FACS) enables a rapid and efficient platform for the identification and enrichment of high-producing cell populations. Here, we have used the combined strategy of FACS-based screening and UCOE to accelerate the clonal selection and enhance recombinant Darbepoetin alfa (DPO) productivity in Chinese Hamster Ovary (CHO) cells. Accordingly, two plasmids were designed: pOptiVEC™ (non-UCOE) and CET1019HD (containing a UCOE). Both contained a codon-optimized Darbepoetin alfa-LoxP-IRES-EGFP-LoxP-IRES-DHFR fragment. To achieve a stable cell line, the cassettes were linearized and transfected into CHO DG44 cells. Then, EGFP was used as a selection marker in FACS to enrich cells with the brightest green fluorescence intensity. Subsequently, DPO and EGFP expression were assessed at the transcriptional and protein levels using qRT-PCR, Flow cytometry, western blotting, and ELISA. Expression analysis revealed that all UCOE-containing cell pools exhibited higher DPO yield compared with non-UCOE populations. Indeed, FACS sorting and enrichment of UCOE-containing cells led to a clone with more than an eightfold increase in productivity. Moreover, isolating high-producing cells via FACS with a simple gate yielded a 1.5-fold increase in target protein concentration compared with unsorted cells. This study demonstrated that UCOE-mediated transcriptional stabilization establishes a robust expression framework while EGFP-based FACS screening enables efficient enrichment of high-producing cells. Combining them enhances protein yield with potential for further optimization in larger-scale applications.