Macrolide- and tetracycline-adjustable siRNA-mediated gene silencing in mammalian cells using polymerase II-dependent promoter derivatives

RNA interference has emerged as a powerful technology for downregulation of specific genes in cells and animals. We have pioneered macrolide- and tetracycline-adjustable short interfering RNA (siRNA) expression for conditional target gene translation fine-tuning in mammalian/human cell lines based on modified RNA polymerase II promoters. Established macrolide- and tetracycline-dependent transactivators/trans-silencers bound and activated modified target promoters tailored for optimal siRNA expression in response to clinical antibiotics' dosing regimes and modulated desired target genes in Chinese hamster ovary (CHO-K1) and human fibrosarcoma (HT-1080) cells with high precision. Further optimization of adjustable RNA polymerase II-based siRNA-specific promoters as well as their combination with various transmodulators enabled near-perfect regulation configurations in specific cell types. Devoid of major genetic constraints compared to basic RNA polymerase III-based siRNA-specific promoters, we expect RNA polymerase II counterparts to significantly advance siRNA-based molecular interventions in biopharmaceutical manufacturing and gene-function analysis as well as gene therapy and tissue engineering.     

Versatile macrolide-responsive mammalian expression vectors for multiregulated multigene metabolic engineering

The novel macrolide-inducible and -repressible mammalian gene regulation systems (E.REX) have been cloned into a variety of sophisticated expression configurations including (1) multi-purpose expression vectors, (2) pTRIDENT-based artificial operons, (3) dual-regulated expression strategies for independent control of two different transgenes, (4) autoregulated vectors for one-step installation of adjustable multigene expression, and (5) oncoretroviral and lentiviral plasmids for transduction of macrolide-, streptogramin- and tetracycline-dependent transactivators and production of cell lines supporting independent control of three different transgenes. This vector portfolio represents a construction kit-like toolbox for efficient installation of adjustable gene expression responsive to clinically licensed antibiotics and enables the design of multiregulated multigene metabolic engineering strategies required for biopharmaceutical manufacturing, gene therapy, and tissue engineering.     

Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells

Background

The recently developed heterologous macrolide‐ (E.REX system) and streptogramin‐ (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines.

Methods

In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic‐dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter.

Results

These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression.

Conclusions

Due to their modular set‐up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell‐specific key regulation components. Copyright © 2002 John Wiley & Sons, Ltd.

     

Multi-gene engineering: simultaneous expression and knockdown of six genes off a single platform

Increases in our understanding of gene function have greatly expanded the repertoire of possible genetic interventions at our disposal with the consequence that many genetic engineering applications require multiple manipulations in which target genes can be both overexpressed and silenced in a simple and co-ordinated manner. Using synthetic introns as a source of encoding short-interfering RNA (siRNA), we demonstrate that it is possible to simultaneously express both a transgene and siRNA from a single polymerase (Pol) II promoter. By encoding siRNA as an intron between two protein domains requiring successful splicing for functionality, it was possible to demonstrate that splicing was occurring, that the coding genes (exonic transgenes) resulted in functional protein, and that the spliced siRNA-containing lariat was capable of modulating expression of a separate target gene. We subsequently extended this concept to develop pTRIDENT-based multi-cistronic vectors that were capable of co-ordinated expression of up to three siRNAs and three transgenes off a single genetic platform. Such multi-gene engineering technology, enabling concomitant transgene overexpression and target gene knockdown, should be useful for therapeutic, biopharmaceutical production, and basic research applications.