Engineering and validation of a dual luciferase reporter system for quantitative and systematic assessment of regulatory sequences in Chinese hamster ovary cells.

Ongoing research efforts to identify potent regulatory sequences that deliver robust and sustained transgene expression are critical for Chinese hamster ovary (CHO) cell line development technologies to meet the growing demand for recombinant proteins. Here we report the engineering and validation of a highly customizable single vector toolkit that comprises an all-in-one dual luciferase reporter system for quantitative and systematic interrogation of transcriptional regulatory sequences in transient and stable transfectants of CHO cells. To model the execution of the reporter system, we implemented a battery of known constitutive promoters including human CMV-mIE, SV40, HSV-TK, mouse PGK, human EF1α, EF1α short (EFS), human UBC, synthetic CAG, and Chinese hamster EF1α (CHEF1α). Of the nine promoters, CMV-mIE yielded the highest transcriptional activity in transient transfection settings, while CHEF1α was the strongest among a select subset of promoters in stable transfectants of CHO-DG44 pools. Remodeling the vector toolkit to build a dual fluorescent reporter system featured an alternative to bioluminescence based reporters. We infer that the findings of this study may serve as a basis to establish new vectors with weak or strong constitutive promoters. Furthermore, the modular all-in-one architecture of the reporter system proved to be a viable tool for discovering novel regulatory sequences that ensure high levels of transient and stable transgene expression in CHO and perhaps other mammalian cell lines.