Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch

Abstract

Applications of conditional gene expression, whether for therapeutic or basic research purposes, are increasingly requiring mammalian gene control systems that exhibit far tighter control properties. While numerous approaches have been used to improve the widely used Tet-regulatory system, many applications, particularly with respect to the engineering of synthetic gene networks, will require a broader range of tightly performing gene control systems. Here, a generically applicable approach is described that utilizes intronically encoded siRNA on the relevant transregulator construct, and siRNA sequence-specific tags on the reporter construct, to minimize basal gene activity in the off-state of a range of common gene control systems. To demonstrate tight control of residual expression the approach was successfully used to conditionally express the toxic proteins RipDD and Linamarase. The intronic siRNA concept was also extended to create a new generation of compact, single-vector, autoinducible siRNA vectors. Finally, using improved regulation systems a mammalian epigenetic toggle switch was engineered that exhibited superior in vitro and in vivo induction characteristics in mice compared to the equivalent non-intronic system.

Control of synthetic gene networks and its applications

“…(C) Schematic diagram for two representative logic gates, the XOR and AND gates reviewed by Singh et al [75]. demonstrated in multiple organisms [18,24,76], and examples of similar topologies are rife in nature [68]. A system can also express multistable behavior through autoactivation [74].…”

Application of Synthetic Biology to Regenerative Medicine

“…These circuits are particularly useful to program mammalian processes with binary characteristics such as differentiation or apoptosis. This transcriptional switch was later improved and used in living mice [40], demonstrating its potential for precise long-term protein delivery in the absence of a sustained input, a critical feature when long term drug administration is potentially toxic or expensive.…”

A New Approach to an Old Problem: Synthetic Biology Tools for Human Disease and Metabolism

“…Bioengineers have used RNAi to tighten control and tweak the expression dynamics of synthetic systems. Greber et al (2008) demonstrated that intronic short interfering RNAs (siRNAs) can reduce basal gene expression of inducible transcription systems (Greber et al 2008). Deans et al (2007) designed an inducible switch in mammalian cells that produces shRNAs to prevent leaky expression of transgenes (Fig.…”