Advertisement

Engineering Elements for Gene Silencing: The Artificial MicroRNAs Technology

  • Pablo Andrés ManavellaEmail author
  • Ignacio Rubio-Somoza
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 732)

Abstract

Small RNA (sRNA)-mediated gene silencing constitutes a powerful tool for the molecular characterization­ of a given gene. The RNAi technology has been largely used for this purpose. This approach is based on the cloning of an inverted repeated fragment of the gene to be silenced. Even when this approach ­produces a strong repression of the target gene it also involves the production of multiple small RNAs species that can easily lead to off targeting. Taking advantage of the latest insights into the new post-biogenesis layer of regulation in microRNA (miRNA) activity, it is possible to overcome the above-mentioned limitation. Artificial microRNAs (amiRNAs) are 21mer small RNAs, which can be genetically engineered and they function to specifically silence single or multiple genes of interest. Since generally just one miRNA molecule is generated from each precursor, the specificity of this technology is much higher than longer inverted repeats. Application of this technology results in highly specific mRNA downregulation by computationally designed sequences programmed to target one or a set of custom-selected transcripts.

Key words

MicroRNA Small RNA attenuation Specific gene downregulation Gene silencing 

Notes

Acknowledgments

The authors would like to thank Proffesor Detlef Weigel and the microRNA team at his lab for continuous and helpful discussion and Beth Rowans for manuscript comments and text editing. Authors are supported by the European Community FP6 IP SIROCCO (contract LSHG-CT-2006-037900) and by the Max Planck Society.

References

  1. 1.
    Voinnet, O. (2009) Origin, biogenesis, and activity of plant microRNAs. Cell. 136, 669–687.PubMedCrossRefGoogle Scholar
  2. 2.
    Lanet, E., Delannoy, E., Sormani, R  ., Floris, M., Brodersen, P., Crété, P., Voinnet, O., Robaglia, C. (2009) Biochemical Evidence for Translational Repression by Arabidopsis MicroRNAs. Plant Cell. 21, 1762–1768.PubMedCrossRefGoogle Scholar
  3. 3.
    Schwab, R., Ossowski, S., Riester, M., Warthmann, N., and Weigel, D. (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell. 18, 1121–1133.PubMedCrossRefGoogle Scholar
  4. 4.
    Ossowski, S., Schwab, R., and Weigel, D. (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J. 53, 674–690.PubMedCrossRefGoogle Scholar
  5. 5.
    Warthmann, N., Chen, H., Ossowski, S., Weigel, D., and Herve, P. (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS One. 3, e1829.PubMedCrossRefGoogle Scholar
  6. 6.
    Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., and Frank, W. (2008) Specific gene silencing by artificial MicroRNAs in Physcomitrella patens: an alternative to targeted gene knockouts. Plant Physiol. 148, 684–693.PubMedCrossRefGoogle Scholar
  7. 7.
    Molnar, A., Bassett, A., Thuenemann, E., Schwach, F., Karkare, S., Ossowski, S., et al. (2009) Highly specific gene silencing by ­artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J. 58, 165–174.CrossRefGoogle Scholar
  8. 8.
    Niu, Q.W., Lin, S.S., Reyes, J.L., Chen, K.C., Wu, H.W., Yeh, S.D., et al. (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat. Biotech. 24, 1420–1428.CrossRefGoogle Scholar
  9. 9.
    Duan, C.G., Wang, C.H., Fang, R.X., and Guo, H.S. (2008) Artificial MicroRNAs Highly Accessible to Targets Confer Efficient Virus Resistance in Plants. J. Virol. 82, 11084–11095.PubMedCrossRefGoogle Scholar
  10. 10.
    Alvarez, J.P., Pekker, I., Goldshmidt, A., Blum, E., Amsellem, Z., and Eshed, Y. (2006) Endogenous and Synthetic MicroRNAs Stimulate Simultaneous, Efficient, and Localized Regulation of Multiple Targets in Diverse Species. Plant cell. 18, 1134–1151.PubMedCrossRefGoogle Scholar
  11. 11.
    Montgomery, T.A., Howell, M.D., Cuperus, J.T., Li, D., Hansen, J.E., Alexander, A.L., et al. (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell. 133, 128–141.PubMedCrossRefGoogle Scholar
  12. 12.
    Khvorova, A., Reynolds, A., and Jayasena, S.D. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell. 115, 209–216.PubMedCrossRefGoogle Scholar
  13. 13.
    Schwarz, D.S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P.D. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell. 115, 199–208.PubMedCrossRefGoogle Scholar
  14. 14.
    Weigel, D., and Glazebrook, J. (2002) Arabidopsis: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 354 pGoogle Scholar
  15. 15.
    Palatnik, J.F., Allen, E., Wu, X., Schommer, C., Schwab, R., Carrington, J.C., et al. (2003) Control of leaf morphogenesis by microRNAs. Nature. 425, 257–263.PubMedCrossRefGoogle Scholar
  16. 16.
    Franco-Zorrilla, J.M., Valli, A., Tudesco, M., Mateos, I., Puga, M.I., Rubio-Somoza, I., et al. (2007) Target mimicry provides a new mechanism for regulation of microRNA ­activity. Nat Genet 39, 1033–1037.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  1. 1.Max Planck Institute for Developmental BiologyTübingenGermany

Personalised recommendations