Summary
The nematode Caenorhabditis elegans shows a high degree of conservation of molecular pathways related to human disease, yet is only 1-mm long and can be considered as a microorganism. Because of the development of a simple but systematic RNA-interference (RNAi) methodology, C. elegans is the only metazoan in which the impact of “knocking-down” nearly every gene in the genome can be analyzed in a whole living animal. Both functional genomic studies and chemical screens can be carried out using C. elegans in vivo screens in a context that preserves intact cell-to-cell communication, neuroendocrine signaling, and every aspect of the animal’s metabolism necessary to survive and reproduce in lab conditions. This feature enables studies that are impossible to undertake in cell-culture-based screens. Although genome-wide RNAi screens and limited small-molecule screens have been successfully performed in C. elegans, they are typically extremely labor-intensive. Furthermore, technical limitations have precluded quantitative measurements and time-resolved analyses.
In this chapter, we provide detailed protocols to carry out automated high-throughput whole-animal RNAi and chemical screens. We describe methods to perform screens in solid and liquid media, in 96 and 384-well format, respectively. We describe the use of automated handling, sorting, and microscopy of worms. Finally, we give information about worm-adapted image analysis tools to quantify phenotypes. The technology presented here facilitates large-scale C. elegans genetic and chemical screens and it is expected to help shed light on relevant biological areas.
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References
Levitan D, Greenwald I. Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer’s disease gene. Nature 1995;377(6547):351–354.
Ogg S, Paradis S, Gottlieb S, et al The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 1997;389(6654):994–999.
Ranganathan R, Sawin ER, Trent C, Horvitz HR. Mutations in the Caenorhabditis elegans serotonin reuptake transporter MOD-5 reveal serotonin-dependent and -independent activities of fluoxetine. J Neurosci 2001;21(16):5871–5884.
Lakso M, Vartiainen S, Moilanen AM, et al. Dopaminergic neuronal loss and motor deficits in Caenorhabditis elegans overexpressing human alpha-synuclein. J Neurochem 2003;86(1):165–172.
Bergamaschi D, Samuels Y, O’Neil NJ, et al. iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human. Nat Genet 2003;33(2):162–167.
Ashrafi K, Chang FY, Watts JL, et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 2003;421(6920):268–272.
Cronin CJ, Feng Z, Schafer WR. Automated imaging of C. elegans behavior. Methods Mol Biol 2006;351:241–251.
Kamath RS, Fraser AG, Dong Y, et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 2003;421(6920):231–237.
Timmons L, Fire A. Specific interference by ingested dsRNA. Nature 1998;395(6705):854.
Lee SS, Lee RY, Fraser AG, Kamath RS, Ahringer J, Ruvkun G. A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat Genet 2003;33(1):40–48.
Frand AR, Russel S, Ruvkun G. Functional genomic analysis of C. elegans molting. PLoS Biol 2005;3(10):e312.
Kim JK, Gabel HW, Kamath RS, et al Functional genomic analysis of RNA interference in C. elegans. Science 2005;308(5725):1164–1167.
Lehner B, Crombie C, Tischler J, Fortunato A, Fraser AG. Systematic mapping of genetic interactions in Caenorhabditis elegans identifies common modifiers of diverse signaling pathways. Nat Genet 2006;38(8):896–903.
Kwok TC, Ricker N, Fraser R, et al A small-molecule screen in C. elegans yields a new calcium channel antagonist. Nature 2006;441(7089):91–95.
Moy TI, Ball AR, Anklesaria Z, Casadei G, Lewis K, Ausubel FM. Identification of novel antimicrobials using a live-animal infection model. Proc Natl Acad Sci U S A 2006;103(27):10414–10419.
Simmer F, Tijsterman M, Parrish S, et al Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Curr Biol 2002;12(15):1317–1319.
Kim DH, Feinbaum R, Alloing G, et al. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity. Science 2002;297(5581):623–626.
Shankar N, Coburn P, Pillar C, Haas W, Gilmore M. Enterococcal cytolysin: activities and association with other virulence traits in a pathogenicity island. Int J Med Microbiol 2004;293(7–8):609–618.
Beanan MJ, Strome S. Characterization of a germ-line proliferation mutation in C. elegans. Development 1992;116(3):755–766.
Pulak R. Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system. Methods Mol Biol 2006;351:275–286.
Acknowledgments
The authors thank Fred Ausubel, Harrison Gabel, and Jonah Larkins-Ford for critical review of the manuscript. We also thank Nicola Tolliday, Bridget Wagner, Lynn Verplank, Jason Burbank, and Anne Carpenter for intellectual and practical contributions to the development of the assays. This work was supported by a grant from the SPARC Program (Broad Institute of Harvard and MIT) to Gary Ruvkun, Fred Ausubel, Eyleen O’Rourke, David Sabatini, and Jonathan Clardy, and by NIH grant AI072508, Fred Ausubel PI.
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O’Rourke, E.J., Conery, A.L., Moy, T.I. (2009). Whole-Animal High-Throughput Screens: The C. elegans Model. In: Clemons, P., Tolliday, N., Wagner, B. (eds) Cell-Based Assays for High-Throughput Screening. Methods in Molecular Biology, vol 486. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-545-3_5
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DOI: https://doi.org/10.1007/978-1-60327-545-3_5
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