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Mammalian shRNA Tools for RNAi

RNA interference (RNAi) is an RNA-mediated gene silencing mechanism. It is a useful tool to investigate the roles of specific genes or to knockdown genes with potentially harmful mutations in therapeutic settings. As a tool in mammalian cell systems, silencing is achieved through the delivery of a double-stranded RNA (dsRNA) that matches the mRNA target sequence. The dsRNA can be delivered as shRNA (short hairpin RNA) or siRNA (short interfering RNA). Delivery can occur via transfection or viral delivery of a plasmid.

shRNAs are often delivered using viral vectors. Lentiviral vectors are a preferred method for long-term gene silencing. Once the viral vector is taken up by a host, the viral genome is transcribed, producing the shRNA. Dicer, an RNase III enzyme, binds the shRNA and cleaves it to produce siRNA. The siRNA then binds the RNA-induced silencing complex (RISC). RISC unwinds the double-stranded siRNA and degrades one of the strands. The remaining single-stranded siRNA then binds the target sequence and RISC cleaves and degrades the mRNA. RISC includes argonaute protein 2 (Ago2), which facilitates cleavage of the target.

RNAi using siRNA follows a similar mechanism. Since it is pre-processed, it can bypass the Dicer cleavage step and proceed to binding of RISC.

For more information on shRNA design and delivery, see resources below.

Mechanism of shRNA-mediated RNA interference shown as cartoon schematic. Image includes description of the steps, including: shRNA is delivered in a viral vector; viral DNA is transcribed to produce shRNA; shRNA is bound and processed to siRNA by Dicer; siRNA binds to RISC and is processed to a single strand; siRNA binds the target sequence and RISC cleaves mRNA. The final image shows a cleaved and degraded mRNA strand.
Figure 1: Overview of shRNA-mediated RNA interference. Created with BioRender.com.

shRNA Plasmids

Browse our selection of empty vectors for cloning in shRNAs. Most plasmids are in a lentiviral backbone, with some retroviral and AAV backbone options. Plasmids for constitutive expression as well as those that allow for conditional (Cre-lox) or inducible (Tet) expression are available. To find plasmids containing other RNAi components, like argonaute, please search our full site.

ID Plasmid Vector Type PI Publication

Additional Resources

Addgene Resources

Web Resources

References

  • Carthew, R. W., & Sontheimer, E. J. (2009). Origins and mechanisms of miRNAs and siRNAs. Cell, 136(4), 642–655. PubMed (Link opens in a new window).
  • Dana, H., Chalbatani, G. M., Mahmoodzadeh, H., Karimloo, R., Rezaiean, O., Moradzadeh, A., Mehmandoost, N., Moazzen, F., Mazraeh, A., Marmari, V., Ebrahimi, M., Rashno, M. M., Abadi, S. J., & Gharagouzlo, E. (2017). Molecular mechanisms and biological functions of siRNA. International Journal of Biomedical Science, 13(2), 48–57. PubMed (Link opens in a new window).
  • Moore, C. B., Guthrie, E. H., Huang, M. T., Taxman, D. J. (2010). Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods in Molecular Biology, 629, 141–158. PubMed (Link opens in a new window).
  • Rao, D. D., Vorhies, J. S., Senzer, N., & Nemunaitis, J. (2009). siRNA vs. shRNA: Similarities and differences. Advanced Drug Delivery Reviews, 61(9), 746–759. PubMed (Link opens in a new window).
  • Wiznerowicz, M., Szulc, J., Trono, D. (2006). Tuning silence: conditional systems for RNA interference. Nature Methods, 3(9), 682–688. PubMed (Link opens in a new window).
  • Zhang, J., Chen, B., Gan, C., Sun, H., Zhang, J., & Feng, L. (2023). A comprehensive review of small interfering RNAs (siRNAs): mechanism, therapeutic targets, and delivery strategies for cancer therapy. International Journal of Nanomedicine, 18, 7605–7635. PubMed (Link opens in a new window).