Furthermore, they may be time-consuming and labor-intensive to implement, making them inefficient tools for quantitative and systematic analysis of known or potentially novel regulatory sequences with robust and stable expression activity in CHO cells.
Despite their widespread utility in mammalian cell cultures, including CHO cells, these systems may inherently suffer from certain limitations such as skewing during normalization, resulting in poor accuracy or experimental bias 12. Normalization is a critical step for mitigating the effects of variables such as cell number, viability, transfection efficiency and DNA copy number, all of which may impose confounding effects on the level of reporter gene expression. Conventional dual vector reporter systems employ two monocistronic vectors and require cotransfection to express the experimental and the internal reporter separately, which serves as a normalization control 11, 12. Genetic reporter systems are powerful molecular tools that can assess the strength of regulatory DNA sequences 10. Based on this premise, the careful selection of transcriptional regulatory sequences that induce robust and stable transgene expression in CHO cells may have a profound effect on the development of effective transgene expression cassettes and vectors. Transcriptional regulatory sequences such as promoters, enhancers, and other cis-acting elements are among the key vector components that significantly impact the level of forced transgene expression, hence the success of an expression vector 7, 9. An optimized architecture and robust genetic elements are imperative to a successful CHO expression vector 7, 8. In this multistep process, a DNA expression vector encoding the foreign protein is initially delivered into CHO cells, followed by a selection process to isolate stable transfectants with integration of expression cassettes into their genome 5, 6. Furthermore, the most intensive research field in recombinant CHO cell line development focuses on establishing clonal cells with robust and stable transgene expression 4. Among these systems, Chinese hamster ovary (CHO) cells remain the primary expression platform 2, 3. At present, mammalian expression systems constitute the most prevalent source of complex multimeric proteins 2. This technology has been widely used in molecular and pharmaceutical biotechnology to engineer host cell lines and produce clinically relevant recombinant proteins such as hormones, enzymes, and monoclonal antibodies 1. The advent of recombinant DNA technology enabled the propagation of genetic material from diverse organisms and their incorporation into a host cell genome. 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. We infer that the findings of this study may serve as a basis to establish new vectors with weak or strong constitutive promoters. Remodeling the vector toolkit to build a dual fluorescent reporter system featured an alternative to bioluminescence based reporters.
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. 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). 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. 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.
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