In the study of gene function, conditional control of gene expression provides improved experimental design and cleaner results than irreversible gene deletion or mutation. However, existing conditional expression systems suffer from inconsistent performance; moreover, they introduce experimental artifacts due to the need for sustained application of small-molecule inducers. The objective of this proposal is to construct a mammalian genetic toggle switch - a conditional expression system in which expression of a transgene can be reversibly switched between stable on and stable off states. The toggle switch will be constructed as a plasmid containing two mutually inhibitory transcription units, each transcription unit consisting of a constitutive promoter and a repressor of the other transcription unit. Because this system will be switched using only transient application of inducing compounds, it will avoid experimental artifacts introduced by existing switches. In addition, the toggle's unique gene-network design will provide for tighter and more consistent control of gene switching.
Application of the toggle switch in cell-based assays and whole animal studies will help biopharmaceutical researchers confirm or refute suspected links between genes and disease and thus accelerate efforts to prioritize potential drug targets for further development.
