Short Hairpin RNA-Mediated Gene Silencing

Abstract

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of shRNA precursors that are effectively processed by the RNAi pathway can lead to potent gene knockdown. The past decade has seen continual advancement and improvement to the various strategies that can be used for shRNA delivery, and the use of shRNAs for clinical applications is well underway. Driving these developments has been the many benefits afforded by shRNA technologies, including the stable integration of expression constructs for long-term expression, infection of difficult-to-target cell lines and tissues using viral vectors, and the temporal control of shRNA transcription by inducible promoters. The use of different effector molecule formats, promoters, and vector types, has meant that experiments can be tailored to target specific cell types and minimize cellular toxicities. Through the application of combinatorial RNAi (co-RNAi), multiple shRNA delivery strategies can improve gene knockdown, permit multiple transcripts to be targeted simultaneously, and curtail the emergence of viral escape mutants. This chapter reviews the history, cellular processing, and various applications of shRNAs in mammalian systems, including options for effector molecule design, vector and promoter types, and methods for multiple shRNA delivery.

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

“…48 The shRNAs can be designed to exhibit promoter-specific expression, 49 and can be genetically integrated to generate stable RNAi regulators. 50 The use of plasmid-based shRNA for gene suppression has been successfully applied in a variety of mammalian cell systems, [51][52][53][54] and T7RNAp-driven shRNAs have been reported for the regulation of gene expression in live mammalian cells 55,56 and zebrafish embryos. 57 Here, we coupled our developed photochemical gene regulation system with an Eg5 shRNA component as a proof of principle study in the photochemical regulation of RNA interference.…”

Effective Inhibition of HIV-1 Production by Short Hairpin RNAs and Small Interfering RNAs Targeting a Highly Conserved Site in HIV-1 Gag RNA Is Optimized by Evaluating Alternative Length Formats

“…shRNA transcripts are composed of two complementary strands that form a stem, separated by a short sequence that forms a loop. They are similar in structure to pre-miRNAs, except that the intended antisense strand is perfectly complementary to its target RNA and the corresponding sense strand in the shRNA stem (27). shRNAs are typically expressed from RNA polymerase III type 3 promoters, which allow for a defined transcript with 2 to 4 uridines at the 3= end from the polymerase termination signal (28,29).…”

Efficient delivery of connective tissue growth factor shRNA using PAMAM nanoparticles

“…Short hairpin RNA (shRNA) is widely used for targeted gene silencing through RNA interference (RNAi) (Lambeth and Smith, 2013). After its discovery in 1998, numerous therapeutic applications of RNAi in biomedical research have been reported, such as for treating human immunodeficiency virus, viral hepatitis, and several other diseases (Ambesajir et al, 2012).…”