Abstract
An ecdysone receptor (EcR)-based inducible gene regulation system that functions in mammalian cells in a tightly regulated fashion is described. This gene switch functions efficiently in a two-hybrid format in which heterologous DNA binding and transcription activation domains are fused to the two heterodimeric receptor partners EcR and RXR. Extensive mutagenesis of the ecdysone receptor along with the development and screening of a large number of non-steroid ligands has resulted in the discovery of several orthogonal ligand-receptor pairs. Receptor mutants were obtained that are activated at low nanomolar concentrations of the ligand. The different components of the gene switch were assembled into a two-vector format or into a single cassette all-in-one vector for expression of genes of interest. Different ecdysone receptor mutants were fused to different DNA binding domains to create several independently acting gene switch combinations (multiplex). The orthogonal nature of the ligand-receptor pairs was demonstrated in reporter assays of two-channel gene switch combinations. Experiments will be presented to demonstrate that the multiplex gene switch system works independently in single cells in response to different ligands. The utility of this system in gene therapy applications will be discussed.