Advertisement

Using RNA Interference to Knock Down the Adhesion Protein TES

  • Elen Griffith
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 370)

Abstract

RNA interference (RNAi) is a specific and efficient method to knock down protein levels using small interfering RNAs (siRNAs), which target mRNA degradation. RNAi can be used in mammalian cell culture systems to target any protein of interest, and several studies have used this method to knock down adhesion proteins. We used siRNAs to knock down the levels of TES, a focal adhesion protein, in HeLa cells. We demonstrated knockdown of both TES mRNA and TES protein. Although total knockdown of TES was not achieved, the observed reduction in TES protein was sufficient to result in a cellular phenotype of reduced actin stress fibers.

Key Words

TES RNAi focal adhesion actin stress fibers HeLa 

References

  1. 1.
    Tobias, E. S., Hurlstone, A. F. L., MacKenzie, E., and Black, D. M. (2001) The TES gene at 7q31.1 is methylated in tumors and encodes a novel growth-suppressing LIM domain protein. Oncogene 20, 2844–2853.PubMedCrossRefGoogle Scholar
  2. 2.
    Coutts, A. S., MacKenzie, E., Griffith, E., and Black, D. M. (2003) TES is a novel focal adhesion protein with a role in cell spreading. J. Cell Sci. 116, 897–906.PubMedCrossRefGoogle Scholar
  3. 3.
    Garvalov, B. K., Higgins, T. E., Sutherland, J. D., et al. (2003) The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions. J. Cell Biol. 161, 33–39.PubMedCrossRefGoogle Scholar
  4. 4.
    Griffith, E., Coutts, A. S., and Black, D. M. (2004) Characterisation of chicken TES and its role in cell spreading and motility. Cell Motil. Cytoskeleton 57, 133–142.PubMedCrossRefGoogle Scholar
  5. 5.
    Griffith, E., Coutts, A. S., and Black, D. M. (2005) RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fiber organisation. Cell Motil. Cytoskeleton 60, 140–152.PubMedCrossRefGoogle Scholar
  6. 6.
    Meister, G. and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349.PubMedCrossRefGoogle Scholar
  7. 7.
    Huppi, K., Martin, S. E., and Caplen, N. J. (2005) Defining and assaying RNAi in mammalian cells. Mol. Cell 17, 1–10.PubMedCrossRefGoogle Scholar
  8. 8.
    Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T., and Weber, K. (2001) Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Sci. 114, 4557–4565.PubMedGoogle Scholar
  9. 9.
    Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Elen Griffith
    • 1
  1. 1.MRC Human Genetics UnitWestern General HospitalEdinburghUK

Personalised recommendations