pHUSH: a single vector system for conditional gene expression

Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer.We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.The development of RNA interference (RNAi) as a tool for reverse genetic studies in mammalian systems has rapidly matured. After the seminal observation that 21 nucleotide, chemically-synthesized RNA duplexes (referred to as short-interfering RNA or siRNA) are capable of targeted gene silencing in mammalian cells [1], RNAi has quickly become a standard technique for functional genetic analysis. A significant advancement of this technique was the development of short-hairpin RNA (shRNA) expression technology [2,3]. This strategy exploits the defined transcriptional start and termination signals of RNA polymerase III (Pol III) promoters to produce a short, inverted transcript. These stem-loop RNA transcripts are then processed within the cell into functional siRNAs and thereby provide a means for the stable suppression of target genes. To this end, multiple groups have reported success in long-term silencing of target genes in engineered cell lines and mice [4,5].Nevertheless, several limitations to the current approach remain. The primary limitation to vector-based shRNA is posed by constitutive hairpin expression. If the shRNA is directed against a gene essential to cell growth and survival, the probability of obtaining a stable line is low, and in those cell lines