Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters

• DOI: 10.1021/acssynbio.0c00643
• Publication Date: 2021-05-03T17:35:59Z
• Authors: Yash D. Patel; Adam J. Brown; Jie Zhu; Guglielmo Rosignoli; Suzanne J. Gibson; Diane Hatton; David C. James
• Keywords: Transcriptional Activation; 0301 basic medicine; 0303 health sciences; Genetic Vectors; Green Fluorescent Proteins; Gene Expression; CHO Cells; Luminescent Proteins; 03 medical and health sciences; Cricetulus; Genes, Reporter; Multigene Family; Animals; Promoter Regions, Genetic; Cell Engineering; Gene Library; Plasmids; Red Fluorescent Protein

To successfully engineer mammalian cells for a desired purpose, multiple recombinant genes are required to be coexpressed at a specific and optimal ratio. In this study, we hypothesized that synthetic promoters varying in transcriptional activity could be used to create single multigene expression vectors coexpressing recombinant genes at a predictable relative stoichiometry. A library of 27 multigene constructs was created comprising three discrete fluorescent reporter gene transcriptional units in fixed series, each under the control of either a relatively low, medium, or high transcriptional strength synthetic promoter in every possible combination. Expression of each reporter gene was determined by absolute quantitation qRT-PCR in CHO cells. The synthetic promoters did generally function as designed within a multigene vector context; however, significant divergences from predicted promoter-mediated transcriptional activity were observed. First, expression of all three genes within a multigene vector was repressed at varying levels relative to coexpression of identical reporter genes on separate single gene vectors at equivalent gene copies. Second, gene positional effects were evident across all constructs where expression of the reporter genes in positions 2 and 3 was generally reduced relative to position 1. Finally, after accounting for general repression, synthetic promoter transcriptional activity within a local multigene vector format deviated from that expected. Taken together, our data reveal that mammalian synthetic promoters can be employed in vectors to mediate expression of multiple genes at predictable relative stoichiometries. However, empirical validation of functional performance is a necessary prerequisite, as vector and promoter design features can significantly impact performance.