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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.

SUBMITTER: Patel YD

PROVIDER: S-EPMC8296667 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression.ResultsA panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants.ConclusionsThe design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

Project description:In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult-to-transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit-like self-inactivating lentiviral expression vector family which is compatible to state-of-the-art packaging and pseudotyping technologies and contains, besides essential cis-acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate-early promoter (P(hCMV)) or the human elongation factor 1alpha (P(hEF1)(alpha)), (iii) P(hCMV-) or P(PGK-) (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein-based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size-optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5'U3, R-U5-psi+-SD, cPPT-RRE-SA and 3'LTR(DeltaU3) modules or placed within the 5'U3 (-78 bp) and 3'LTR(DeltaU3) (8666 bp). pLentiModule enables straightforward cassette-type module swapping between lentiviral expression vector family members and facilitates the design of Tat-independent (replacement of 5'LTR by heterologous promoter elements), regulated and self-excisable proviruses (insertion of responsive operators or LoxP in the 3'LTR(DeltaU3) element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi-purpose construction kit-like vector series described here is compatible with itself as well as many other (non-viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human diseases.

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Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters.

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