A Novel Mammalian Gene Regulation System Responsive to Streptogramin Antibiotics

Abstract

We designed a new strategy for efficient regulation of cloned gene expression in mammalian cells. The new gene regulation system is responsive to streptogramins, a class of human therapy-proven antibiotics such as pristinamycin and virginiamycin, which are naturally produced by Streptomyces spp. The pristinamycin-responsive interaction between the repressor (Pip) of the pristinamycin resistance gene (ptr) and its target sequence the ptr promoter (P-PTR) formed the basis for construction of two chimeric genetic determinants: Pip was fused to a eukaryotic transactivator domain (PIT, pristinamycin-responsive transactivator) and the Pip binding DNA sequence (P-PTR) was cloned 5' of a minimal insect promoter resulting in P-PIR (pristinamycin I-responsive promoter). PIT retains its streptogramin-dependent P-PTR binding capacity in mammalian cells which enables sufficiently close contact between the transactivation domain and the minimal promoter to achieve transcription initiation in absence of streptogramin antibiotics. In the presence of streptogramins, PIT is released from P-PIR and gene expression is abolished. The novel PIT/P-PIR system shows excellent regulation characteristics (high expression levels and low basal activity) in a variety of mammalian cell lines including CHO-K1, BHK-21 and HeLa cells. In addition, the streptogramin regulation system is compatible with the well-established tetracycline-responsive expression concept which enables the combined use of these two distinct systems in a single cell for independent control of different transgenes.