Mechanism of Action
A small hairpin RNA or short hairpin RNA(shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). shRNA are synthesized in the nucleus of cells, further processed and transported to the cytoplasm and then incorporated into the RNA-interfering silencing complex (RISC) for activity. Expression of shRNA in cells can incorporate different promoters and is accomplished by delivery of plasmids or through viral or bacterial vectors. For additional information, please view a video by Greg Hannon of CSHL.
What is shRNA?
Expression of shRNA in cells can be obtained by delivery of plasmids or through viral or bacterial vectors, which provides flexibility in the design of the vector. Delivery of plasmids to cells through transfections to obtain shRNA expression can be accomplished utilizing commercial reagents in vitro. Most vector-based shRNA systems contain a selectable marker to allow for the elimination of cells that have not been successfully transfected or transduced, and maintenance of cells with sustained gene knockdown. The shRNA expression cassettes can also be incorporated into viral vector systems, including retrovirus, adeno-associated virus, adenovirus and lentivirus, which permit stable integration into and expression from the host genome. These viral strategies permit shRNA delivery to cell lines that are refractory to transfection. Fluorescent markers (such as a Green or Red Fluorescent Protein [GFP or RFP]) can also be included for tracking cells expressing shRNAs. The performance of shRNA is influenced by many factors including the efficiency of transduction or transfection, the promoter driving expression of the shRNA and epigenetic modifications (which can lead to silencing of shRNA expression). Further, the influence that each of these factors have on vector performance can differ depending on the cell line or cell type.
How is shRNA delivered to a cell?
Once the vector has integrated into the host genome, the shRNA is then transcribed in the nucleus through different promoters such as polymerase II or polymerase III. These initial precursors mimics pri-microRNA (pri-miRNA) and are processed by Drosha and its dsRNA-binding partner DGCR8. The resulting pre-shRNA is exported from the nucleus by Exportin 5. The pre-shRNA is then cleaved by Dicer and TRBP/PACT, removing the hairpin and creating a 20–25 nt double-stranded siRNA with 2 nt 3’ overhangs at each end. The sense (passenger) strand is degraded. The antisense (guide) strand directs RISC to mRNA that has a complementary sequence. In the case of perfect complementarily, RISC cleaves the mRNA. In the case of imperfect complementarily, RISC represses translation of the mRNA. In both of these cases, the shRNA leads to target gene silencing.
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