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cDNA Libraries for Virus-Induced Gene Silencing

  • Andrea T. Todd
  • Enwu Liu
  • Jonathan E. Page
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 631)

Abstract

Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput functional genomics in plants. Combined with the use of subtractive cDNA libraries for generating a collection of VIGS-ready cDNA inserts, VIGS can be utilized to screen a large number of genes to determine phenotypes resulting from the knockdown/knockout of gene function. Taking into account the optimal insert design for VIGS, we describe a methodology for producing VIGS-ready cDNA libraries enriched for inserts relevant to the biological process of interest.

Key words

Functional genomics RNA silencing Virus-induced gene silencing VIGS siRNAs Plant viruses Tobravirus Tobacco rattle virus Nicotiana benthamiana 

Notes

Acknowledgments

We are grateful to S. Dinesh-Kumar (Yale University) for generously providing the TRV vectors. We also thank the DNA Service Unit at NRC-PBI for EST sequencing, Jacek Nowak and Kannan Vijayan for bioinformatic analysis, and Sandra Polvi for assistance in plant cultivation. This is manuscript NRCC #50134.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Andrea T. Todd
    • 1
  • Enwu Liu
    • 1
  • Jonathan E. Page
    • 1
  1. 1.NRC Plant Biotechnology InstituteSaskatoonCanada

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