Abstract
BackgroundConditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector.ResultsBoth 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.ConclusionWe 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.
Highlights
Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies
The entire Melk cDNA was cloned downstream of the pGL3-Luc stop codon, and the resulting pGL3-Melk construct was used as a reporter for short-hairpin RNA (shRNA) efficacy in transient co-transfection experiments
We first compared several published strategies for shRNA design based on two target sequences: sh7 and shB
Summary
Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. 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. BMC Biotechnology 2007, 7:61 http://www.biomedcentral.com/1472-6750/7/61 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 processed within the cell into functional siRNAs and thereby provide a means for the stable suppression of target genes. Multiple groups have reported success in long-term silencing of target genes in engineered cell lines and mice [4,5]
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