1033. Tightly Controlled and Reversible Knockdown of Gene Expression Using Lentiviral Vectors Encoding Tetracycline-Dependent Repressors or Transactivators

RNAi accomplished in mammalian cells by the introduction of small interfering RNAs (siRNAs) is a powerful emerging tool for reverse genetic studies. To control siRNA-mediated gene knockdown both spatially and temporally, we undertook the development of HIV-1-based lentiviral vector systems for the conditional, doxycycline (Dox) dependent production of short hairpin RNAs (shRNAs) in mammalian cells from modified human RNA polymerase III (Pol III) promoters. Our results showed that expression of shRNAs from engineered human U6 (hU6) promoters containing a single tetracycline operator (TO) sequence between the proximal sequence element (PSE) and the TATA box, or a second- generation Tet-responsive promoter element (TRE) upstream ofthe promoter was tight and reversible in the presence of a modified Tet-repressor (TetR). Reversible repression of shRNA production was mediated by doxycycline (Dox) addition. We also established a novel hU6 promoter system in which the distal sequence element (DSE) of the hU6 promoter was replaced with a second-generation TRE element. In this system, positive regulation of shRNA production is mediated by a novel Tet-dependent transactivator bearing the transactivator domain of the human Sp1 transcription factor. The results obtained using EGFP and human CXCR4 chemokine receptor mRNAs as targets suggest that these vectors systems are useful for tight and controlled gene knockdown in mammalian cells in vitro. They may ultimately find applications in vivo including transgenic mouse models of human disease.