1059. Development of a Novel Regulatory System Allowing Doxycycline-Controlled Suppression of Target Genes in Mammalian Cells

Abstract

Top of pageAbstract The efficient and specific suppression of target genes by RNA interference (RNAi) is a remarkable new approach that may find applications in human therapy whenever genes involved in the respective pathology have to be inhibited. Clinical applications will require a regulatory system that allows control of RNAi by administration of a small inducer molecule. The treatment could then be adapted to the needs of the patient and, should complications arise, the therapy could be stopped or interrupted. RNAi based gene suppression can be induced in target cells by short hairpin RNAs (shRNAs) that are expressed under the control of RNA polymerase III promoters in order to avoid disturbing poly A sequences. To control RNAi by inducible and reversible expression of shRNAs, we developed a novel regulatory system. This system is based on a novel tetracycline-dependent transactivator capable of inducing transcription of shRNAs from a minimal RNA polymerase III promoter in the presence of doxycycline. As a proof-of-principle, tetracycline-controlled RNA interference was used to regulate the expression of GFP in HEK 293T cells stably expressing this transgene. RNA interference was induced by administration of doxycycline yielding a 90% reduction of GFP expression. The effect of doxycycline on the expression of GFP was dose- and time-dependent. In particular, the down-regulation of GFP was reversible after withdrawal of doxycycline, as observed by reappearance of GFP fluorescence.