Abstract
Double-stranded RNA interference (RNAi) is an effective method for disrupting expression of specific genes in Caenorhabditis elegans and other organisms1,2,3,4,5. Applications of this reverse-genetics tool, however, are somewhat restricted in nematodes because introduced dsRNA is not stably inherited5. Another difficulty is that RNAi disruption of late-acting genes has been generally less consistent than that of embryonically expressed genes, perhaps because the concentration of dsRNA becomes lower as cellular division proceeds or as developmental time advances1. In particular, some neuronally expressed genes appear refractory to dsRNA-mediated interference. We sought to extend the applicability of RNAi by in vivo expression of heritable inverted-repeat (IR) genes. We assayed the efficacy of in vivo-driven RNAi in three situations for which heritable, inducible RNAi would be advantageous: (i) production of large numbers of animals deficient for gene activities required for viability or reproduction; (ii) generation of large populations of phenocopy mutants for biochemical analysis; and (iii) effective gene inactivation in the nervous system. We report that heritable IR genes confer potent and specific gene inactivation for each of these applications. We suggest that a similar strategy might be used to test for dsRNA interference effects in higher organisms in which it is feasible to construct transgenic animals, but impossible to directly or transiently introduce high concentrations of dsRNA.
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References
Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).
Montgomery, M.K., Xu, S. & Fire, A. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 95, 15502 –15507 (1998).
Timmons, L. & Fire, A. Specific interference by ingested dsRNA . Nature 395, 854 (1998).
Montgomery, M.K. & Fire, A. Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression. Trends Genet. 14, 255–258 (1998).
Tabara, H., Grishok, A. & Mello, C.C. RNAi in C. elegans—soaking in the genome sequence. Science 282, 430– 431 (1998).
Jones, D., Russnak, R.H., Kay, R.J. & Candido, E.P.M. Structure, expression and evolution of a heat-shock gene locus in C. elegans that is flanked by repetitive elements. J. Biol. Chem. 261 , 12006–12015 (1986).
Stringham, E.G., Dixon, D.K., Jones, D. & Candido, E.P.M. Temporal and spatial expression patterns of the small heat shock (hsp-16) genes in transgenic Caenorhabditis elegans. Mol. Biol. Cell 3, 221–233 (1992).
Fire, A., Harrison, S.W. & Dixon, D. A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene 93 , 189–198 (1990).
Lu, X. & Horvitz, H.R. lin-35 and lin-53, two genes that antagonize a C. elegans Ras pathway, encode proteins similar to Rb and its binding protein RbAp48. Cell 95, 981–991 (1998).
Zhang Y., LeRoy, G., Seelig, H.P., Lane, W.S. & Reinberg, D. The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities. Cell 95, 279–289 (1998).
Ryazanov, A.G. et al. Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc. Natl Acad. Sci. USA 94, 4884–4889 ( 1997).
Maduro, M. & Pilgrim, D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics 141, 977–988 ( 1995).
Ren, X.-C, Kim, S., Fox, E., Hedgecock, E. & Wadsworth, W.G. Role of netrin UNC-6 in patterning the longitudinal nerves of Caenorhabditis elegans. J. Neurobiol. 39, 107–118 (1999).
Mitani, S., Du, H., Hall, D., Driscoll, M. & Chalfie, M. Combinatorial control of touch receptor neuron expression in Caenorhabditis elegans. Development 119, 773–783 (1993).
Park, E.-C & Horvitz, H.R. Mutations with dominant effects on the behavior and morphology of the nematode C. elegans. Genetics 113, 821–852 ( 1986).
Dibb, N.J., Maruyama, I.N., Krause, M. & Karn, J. Sequence analysis of the complete Caenorhabditis elegans myosin heavy chain gene family. J. Mol. Biol. 205, 603– 613 (1989).
Kennerdell, J.R. & Carthew, R.W. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017– 1026 (1998).
Ngo, H., Tschudi, C., Gull, K. & Ullu, E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc. Natl Acad. Sci. USA 95, 14687–14692 (1998).
Voinnet, O., Vain, P., Angell, V. & Baulcombe, D.C. Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA. Cell 95, 177–187 (1998).
Sánchez Alvarado, A. & Newmark, P.A. Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc. Natl Acad. Sci. USA 96, 5049– 5054 (1999).
Waterhouse, P.M., Graham, M.W. & Wang, M.B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. USA 95, 13959– 13964 (1998).
Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).
Mello, C.C. & Fire, A. DNA transformation. in Methods in Cell Biology. Caenorhabditis elegans: Modern Biological Analysis of an Organism (eds Epstein, H.F. & Shakes, D.C. ) 451–482 (Academic Press, San Diego, 1995).
Kramer, J.M., French, R.P., Park, E.-C & Johnson, J.J. The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen. Mol. Cell. Biol. 10, 2081–2089 (1990).
Tabara, H. et al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123– 132 (1999).
Acknowledgements
We thank G. Patterson for critical reading of the manuscript and K. Pavur for eEF-2 kinase assays. This work was supported by grants from the National Institutes of Health (M.D. NS37955, NS344435; A.R. GM57300). N.T. is supported by a Human Frontiers in Science Program Organization Research Fellowship.
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Tavernarakis, N., Wang, S., Dorovkov, M. et al. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat Genet 24, 180–183 (2000). https://doi.org/10.1038/72850
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DOI: https://doi.org/10.1038/72850
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