Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters

Abstract

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.

Engineered Fungus Thermothelomyces thermophilus Producing Plant Storage Proteins

“…The downstream regulatory elements located within CDSs have been previously identified in viruses, yeasts and mammalians [41][42][43]. The gene positional effects were evident during co-expression of fluoresce reporter genes under three heterotypic mammalian synthetic promoters in the positions 2 and 3 that were generally reduced relatively to the position 1 [44]. Moreover, the codon usage may also affect the mRNA levels during heterologous gene expression in Th.…”

“…However, the 5 -end of OpIE2 insect viral promoter was found to be blocking the activity of the CMV promoter, constructed in a tandem arrangement, when transforming mammalian cells [49]. Evidently, the promoter interference cannot always be anticipated and empirical validation of functionality should be a prerequisite, as vector containing more than one target gene and promoters and promoter design features can significantly impact performance [40,44]. Only exchanging the terminators TtrpC for Ttef1 for a bidirectional histone-ge promoters system in Aspergillus resulted in a significant increase in the relative multige expression by a factor of 1.8, while they were interchangeable and equally efficient f many other expression constructs [48].…”

“…Moreover, the use of the similar promoters in simultaneously operating dual-promoter vector system was thought to interfere wi their effectiveness, because the promoter interference has the potential to occur betwe vector-borne promoters and endogenous promoters following vector integration [4 However, the 5′-end of OpIE2 insect viral promoter was found to be blocking the activi of the CMV promoter, constructed in a tandem arrangement, when transforming mam malian cells [49]. Evidently, the promoter interference cannot always be anticipated an empirical validation of functionality should be a prerequisite, as vector containing mo than one target gene and promoters and promoter design features can significantly im pact performance [40,44].…”

“…Such AAs as His, Trp, and Cys were found to tend to be the most rarely used during normal growth and, therefore, the costliest to biosynthesize in E. coli [55]. Given that there are no clear strategies to engineer filamentous fungi for increased secretion of a protein of interest, methods are required that are able to construct and test many different combinations of vectors and promoters, other transcriptional and/or translational control elements, and genome modifications for the high-throughput screening of engineered strains and the correlated scale production [19,44].…”

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells

“…CMV promoters are considered to be one of the strongest viral promoters, and hEF-1α promoters have the stronger transcription initiation efficiency and are more suitable for large-scale production of recombinant antibodies ( Ebadat et al, 2017 ). In recent years, the construction of synthetic or heterozygous promoters will become an effective strategy to improve the transcription efficiency ( Patel et al, 2021 ).…”