Abstract
Hairpin (hpRNA)-mediated gene silencing exploits a cellular mechanism that recognizes double-stranded RNA (dsRNA) and subjects it and its corresponding mRNA to a sequence-specific degradation. This phenomenon is known as posttranscriptional gene silencing (PTGS) in plants and RNA interference (RNAi) in animals. dsRNA, when introduced into plant cells through hpRNA constructs, results in severe reduction of the target mRNA—the silencing effect being stably inherited over many generations. While hpRNA constructs can be made using conventional plasmids, use of generic vectors such as pHANNIBAL makes it more convenient to silence a number of genes simultaneously. Vectors, such as pHELLSGATE, that are based on the Gateway® technology are suitable for high-throughput gene silencing. The specificity of dsRNA silencing, it’s ability to simultaneously silence multiple genes combined with the availability of high-throughput silencing vectors enables the researcher to generate custom knockout plants.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hannon, G. J. (2002) RNA interference. Nature 418, 244–251.
Gonczy, P., Echeverri, C., Oegema, K., et al. (2000) Functional genomic analysis of cell division in C-elegans using RNAi of genes on chromosome III. Nature 408, 331–336.
Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., and Ahringer (2000) Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325–330.
Waterhouse, P. M., Graham, M. W., and Wang, M.-B. (1998) Virus resistance and gene silencing in plants is induced by double-stranded RNA. Proc. Nat. Acad. Sci. USA 95, 13959–13964.
Smith, N. A., Singh, S. P., Wang, M.-B., Stoutjesdijk, P., Green, A., and Waterhouse, P. M. (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407, 319–320.
Wesley, S. V., Helliwell, C. A., Smith, N., et al. (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27, 581–590.
Helliwell, C. A., Wesley, S. V., Wielopolska, A. J., and Waterhouse, P. M. (2002) High throughput vectors for efficient gene silencing in plants. Functional Plant Biol. 29, 1217–1225
Gleave, A. P. (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203–1207.
Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.
Horser, C., Abbott, D. C, Wesley, S. V., Smith, N. A., and Waterhouse, P. M. (2001) Gene silencing—principles and application, in Genetic Engineering, Vol. 23. (Setlow, J., ed.), Kluwer Academic, New York.
Liu, Q., Singh, S., and Green, A. (2002) High-stearic and oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol. 129, 1732–1743.
Chuang, C. F. and Meyerowitz, E. M. (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc. Nat. Acad. Sci. USA 97, 4985–4990.
Levin, J. Z., Framond, A. J., Tuttle, A., Bauer, M. W., and Heifetz, P. B. (2000) Methods of double-stranded RNA-mediated gene inactivation in Arabidopsis and their use to define an essential gene in methionine biosynthesis. Plant Mol. Biol. 44, 759–775.
Wang, M.-B., Abbott D. C., and Waterhouse, P. M. (2000) A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol. Plant Pathol. 1, 347–356.
Stoutjesdijk, P., Singh, S. P., Liu, Q., Hurlstone, C. J., Waterhouse, P. M., and Green, A. G. (2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol. 129, 1723–1731.
Wang, M.-B. and Waterhouse, P. M. (2002) Application of gene silencing in plants. Curr. Opin. Plant. Biol. 5, 146–150.
Klink, V. P. and Wolniak, S. M. (2000) The efficacy of RNAi in the study of the plant cytoskeleton. J. Plant. Growth. Regul. 19, 371–384.
Schweizer, P., Pokorny, J., Schulze-Lefert, P., and Dudler, R. (2000) Double-stranded RNA interferes with gene function at the single-cell level in cereals. Plant J. 24, 895–903.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Wesley, S.V. et al. (2003). Custom Knock-Outs with Hairpin RNA-Mediated Gene Silencing. In: Grotewold, E. (eds) Plant Functional Genomics. Methods in Molecular Biology™, vol 236. Humana Press. https://doi.org/10.1385/1-59259-413-1:273
Download citation
DOI: https://doi.org/10.1385/1-59259-413-1:273
Publisher Name: Humana Press
Print ISBN: 978-1-58829-145-5
Online ISBN: 978-1-59259-413-9
eBook Packages: Springer Protocols