A regulatory system for use in gene transfer.

Abstract

We recently have demonstrated that a C-terminal deletion mutant of the human progesterone receptor (hPRB891) fails to bind to progesterone but can bind RU 486 (Mifepristone) and other progesterone antagonists. Most significantly, this mutant receptor activates transcription of a reporter gene containing the progesterone response element in the presence of these antagonists. Taking advantage of this finding and the modular nature of functional domains of steroid receptors, we constructed a chimeric regulator (pGL-VP) by fusing the ligand-binding domain of human progesterone receptor hPRB891 to the yeast transcriptional activator GAL4 DNA-binding domain and the herpes simplex virus protein VP16 activation domain. We demonstrated that this chimeric regulator activates target genes containing the GAL4-binding sites in transient transfection assays in response to RU 486. In addition, this regulatory system has been validated by ex vivo transplantation of a stable cell line containing both the regulator and a reporter gene into rats. The dosage of RU 486 used is significantly lower than that required for antagonizing progesterone action. The gene-switch system reported here represents a regulatory system, which could be applicable for gene-transfer studies involving animals, as well as humans, in which the delivered gene(s) can be specifically turned on/off in response to an exogenous compound.