Abstract
The regulation of gene expression in mammalian cells by combining various cis-regulatory features has rarely been discussed. In this study, we constructed expression vectors containing various combinations of regulatory elements to examine the regulation of gene expression by different combinations of cis-regulatory elements. The effects of four promoters (CMV promoter, PGK promoter, Polr2a promoter, and EF-1α core promoter), two enhancers (CMV enhancer and SV40 enhancer), two introns (EF-1α intron A and hybrid intron), two terminators (CYC1 terminator and TEF terminator), and their different combinations on downstream gene expression were compared in various mammalian cells using fluorescence microscopy to observe fluorescence, quantitative real-time PCR (qRT-PCR), and western blot. The receptor binding domain (RBD) sequence from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein was used to replace the eGFP sequence in the expression vector and the RBD expression was detected by qRT-PCR and western blot. The results showed that protein expression can be regulated by optimizing the combination of cis-acting elements. The vector with the CMV enhancer, EF-1α core promoter, and TEF terminator was found to express approximately threefold higher eGFP than the unmodified vector in different animal cells, as well as 2.63-fold higher recombinant RBD protein than the original vector in HEK-293T cells. Moreover, we suggest that combinations of multiple regulatory elements capable of regulating gene expression do not necessarily exhibit synergistic effects to enhance expression further. Overall, our findings provide insights into biological applications that require the regulation of gene expression and will help to optimize expression vectors for biosynthesis and other fields. Additionally, we provide valuable insights into the production of RBD proteins, which may aid in developing reagents for diagnosis and treatment during the COVID-19 pandemic.
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All relevant data are within the manuscript and its Supporting Information files. The data and materials generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. We understand the importance of data sharing and are committed to promoting transparency and reproducibility. Interested researchers can contact the corresponding author to request access to the data and materials associated with this study. We will make every effort to respond to data requests promptly and provide the necessary information to facilitate further research and collaboration.
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Acknowledgements
This research was supported by Grants from the Doctoral Foundation of Henan Agricultural University (30501221) and the National Key R&D Program of China (2021YFD1301200).
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Zhou, LY., Zhang, S., Li, LY. et al. Optimization of mammalian expression vector by cis-regulatory element combinations. Mol Genet Genomics 298, 1121–1133 (2023). https://doi.org/10.1007/s00438-023-02042-0
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DOI: https://doi.org/10.1007/s00438-023-02042-0