1061. An Ecdysone Receptor-Based Gene Switch for Precisely and Variably Regulated Expression of Genes in Mammalian Cells

Abstract

Top of pageAbstract Different versions of an ecdysone receptor (EcR)-based gene switch that can be regulated by synthetic non-steroid ligands have been developed and reported previously. The most active ligands that are used for regulating these switch systems are the diacylhydrazine class of compounds that exhibit very favorable toxicological properties in the mammalian cells. The gene switch is designed in a two-hybrid format and consists of two nuclear receptors that heterodimerize to form the transcriptional activator. The receptor that binds the activating ligand is the DEF domain of EcR that is fused to a DNA binding domain (DBD) such as Gal4 (1|[ndash]|147 a.a.). The heterodimerizing partner is the EF domain of a chimeric RXR that is fused to a transcriptional activation domain such as VP16. Ligand binding to the EcR enhances its interaction with its RXR partner and activates transcription from a responsive promoter that contains the binding sites for the DBD-EcR(DEF). The gene of interest that is inserted downstream of the responsive promoter can be regulated in a dose-dependent manner by the ligand. A repertoire of inducible promoters have been incorporated into different versions of the switch. Data will be presented in which the gene switch provides very tight but moderate expression levels for genes that require stringent regulation. At the other end of this spectrum are the inducible promoters allowing slightly leaky but very high expression levels for applications where the absolute expression levels are more important. The switch system was originally designed as a three-vector system in which the two receptor components and the inducible expression elements were on three separate vectors. Presently, the gene switch has been assembled into a single vector system that accommodates all the three components of the switch as a single all-in-one cassette. In the initial all-in-one vector, the two receptor components were expressed from two different constitutive promoters. In the later versions, the receptors are expressed from a single constitutive promoter as a dicistronic message with an IRES sequence in the middle. Modifications in the switch design resulted in an all-in-one cassette that is small enough to be accommodated into viral vectors.