Skip to main content
SpringerLink
Log in

Allele-Specific Silencing by RNA Interference

  • Protocol
  • First Online:
RNA Interference

Part of the book series: Methods in Molecular Biology ((MIMB,volume 623))

Abstract

Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. We have developed an assay system with reporter alleles that encode the Photinus and Renilla luciferase genes carrying mutant and wild-type allelic sequences in their 3′-untranslated regions. The assay system allows us to evaluate designed siRNAs and also short hairpin RNAs for allele-specific silencing against target mutant alleles as well as off-target silencing against corresponding wild-type alleles simultaneously in a qualitative and quantitative manner.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Dykxhoorn, D.M., Novina, C.D. and Sharp, P.A. (2003) Killing the messenger: short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4, 457–467.

    Article  CAS  PubMed  Google Scholar 

  2. Meister, G. and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349.

    Article  CAS  PubMed  Google Scholar 

  3. Victor, M., Bei, Y., Gay, F., Calvo, D., Mello, C. and Shi, Y. (2002) HAT activity is essential for CBP-1-dependent transcription and differentiation in Caenorhabditis elegans. EMBO Rep. 3, 50–55.

    Article  CAS  PubMed  Google Scholar 

  4. Wood, M., Yin, H. and McClorey, G. (2007) Modulating the expression of disease genes with RNA-based therapy. PLoS Genet. 3, e109.

    Article  PubMed  Google Scholar 

  5. Miller, V.M., Gouvion, C.M., Davidson, B.L. and Paulson, H.L. (2004) Targeting Alzheimer’s disease genes with RNA interference: an efficient strategy for silencing mutant alleles. Nucleic Acids Res. 32, 661–668.

    Article  CAS  PubMed  Google Scholar 

  6. Miller, V.M., Xia, H., Marrs, G.L., Gouvion, C.M., Lee, G., Davidson, B.L. and Paulson, H.L. (2003) Allele-specific silencing of dominant disease genes. Proc. Natl. Acad. Sci. U.S.A. 100, 7195–7200.

    Article  CAS  PubMed  Google Scholar 

  7. Ohnishi, Y., Tokunaga, K., Kaneko, K. and Hohjoh, H. (2006) Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles. J. RNAi Gene Silencing, 2, 154–160.

    PubMed  Google Scholar 

  8. Xia, H., Mao, Q., Eliason, S.L., Harper, S.Q., Martins, I.H., Orr, H.T., et al. (2004) RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat. Med. 10, 816–820.

    Article  CAS  PubMed  Google Scholar 

  9. Ohnishi, Y., Tamura, Y., Yoshida, M., Tokunaga, K. and Hohjoh, H. (2008) Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi. PLoS ONE 3, e2248.

    Article  PubMed  Google Scholar 

  10. Rodriguez-Lebron, E. and Paulson, H.L. (2006) Allele-specific RNA interference for neurological disease. Gene Ther. 13, 576–581.

    Article  CAS  PubMed  Google Scholar 

  11. Maxwell, M.M., Pasinelli, P., Kazantsev, A.G. and Brown, R.H., Jr. (2004) RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells. Proc. Natl. Acad. Sci. U.S.A. 101, 3178–3183.

    Article  CAS  PubMed  Google Scholar 

  12. Denovan-Wright, E.M. and Davidson, B.L. (2006) RNAi: a potential therapy for the dominantly inherited nucleotide repeat diseases. Gene Ther. 13, 525–531.

    Article  CAS  PubMed  Google Scholar 

  13. Ohnishi, Y., Tokunaga, K. and Hohjoh, H. (2005) Influence of assembly of siRNA elements into RNA-induced silencing complex by fork-siRNA duplex carrying nucleotide mismatches at the 3′- or 5′-end of the sense-stranded siRNA element. Biochem. Biophys. Res. Commun. 329, 516–521.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The author would like to thank Yusuke Ohnishi and Mariko Yoshida for their helpful assistance. This work was supported by a research grant from the Ministry of Health, Labor, and Welfare of Japan and by a Grant-in-Aid from the Japan Society for the Promotion of Science.

Author information

Authors and Affiliations

  1. National Institute of Neuroscience, NCNP, Tokyo, Japan

    Hirohiko Hohjoh

Authors
  1. Hirohiko Hohjoh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hirohiko Hohjoh .

Editor information

Editors and Affiliations

  1. Dept. Surgery, Pathology & Oncology, University of Western Ontario, Windermere Road 339, London, N6A 5A5, Canada

    Wei-Ping Min

  2. MediStem Laboratories, Inc., E. Cedar Street 2027, Tempe, 85281, USA

    Thomas Ichim

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Hohjoh, H. (2010). Allele-Specific Silencing by RNA Interference. In: Min, WP., Ichim, T. (eds) RNA Interference. Methods in Molecular Biology, vol 623. Humana Press. https://doi.org/10.1007/978-1-60761-588-0_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-588-0_4

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-587-3

  • Online ISBN: 978-1-60761-588-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation