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Genome-Wide RNAi Screens in C. elegans to Identify Genes Influencing Lifespan and Innate Immunity

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High-Throughput RNAi Screening

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1470))

Abstract

RNA interference is a rapid, inexpensive, and highly effective tool used to inhibit gene function. In C. elegans, whole genome screens have been used to identify genes involved with numerous traits including aging and innate immunity. RNAi in C. elegans can be carried out via feeding, soaking, or injection. Here we outline protocols used to maintain, grow, and carry out RNAi via feeding in C. elegans and determine whether the inhibited genes are essential for lifespan or innate immunity.

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References

  1. Corsi AK, Wightman B, Chalfie M (2015) A transparent window into biology: a primer on Caenorhabditis elegans. Genetics 200:387–407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811

    Article  CAS  PubMed  Google Scholar 

  3. Li H, Li WX, Ding SW (2002) Induction and suppression of RNA silencing by an animal virus. Science 296:1319–1321

    Article  CAS  PubMed  Google Scholar 

  4. Ding S-W, Li H, Lu R et al (2004) RNA silencing: a conserved antiviral immunity of plants and animals. Virus Res 102:109–115

    Article  CAS  PubMed  Google Scholar 

  5. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2:279–289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wilson RC, Doudna JA (2013) Molecular mechanisms of RNA interference. Annu Rev Biophys 42:217–239

    Article  CAS  PubMed  Google Scholar 

  7. Timmons L, Fire A (1998) Specific interference by ingested dsRNA. Nature 395:854

    Article  CAS  PubMed  Google Scholar 

  8. Tabara H, Grishok A, Mello CC (1998) RNAi in C. elegans: soaking in the genome sequence. Science 282:430–431

    Article  CAS  PubMed  Google Scholar 

  9. Kamath RS, Fraser AG, Dong Y et al (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421:231–237

    Article  CAS  PubMed  Google Scholar 

  10. Rual J-F, Ceron J, Koreth J et al (2004) Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res 14:2162–2168

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Squiban B, Belougne J, Ewbank J, Zugasti O (2012) Quantitative and automated high-throughput genome-wide RNAi screens in C. elegans. J Vis Exp. doi: 10.3791/3448

  12. Simmer F, Tijsterman M, Parrish S et al (2002) Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Curr Biol 12:1317–1319

    Article  CAS  PubMed  Google Scholar 

  13. Kennedy S, Wang D, Ruvkun G (2004) A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427:645–649

    Article  CAS  PubMed  Google Scholar 

  14. Lehner B, Calixto A, Crombie C et al (2006) Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference. Genome Biol 7:R4. doi:10.1186/gb-2006-7-1-r4

    Article  PubMed  PubMed Central  Google Scholar 

  15. Ashrafi K, Chang FY, Watts JL et al (2003) Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421:268–272

    Article  CAS  PubMed  Google Scholar 

  16. Sönnichsen B, Koski LB, Walsh A et al (2005) Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434:462–469

    Article  PubMed  Google Scholar 

  17. Lee SS, Lee RYN, Fraser AG et al (2003) A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat Genet 33:40–48

    Article  CAS  PubMed  Google Scholar 

  18. Murphy CT, McCarroll SA, Bargmann CI et al (2003) Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424:277–283

    Article  CAS  PubMed  Google Scholar 

  19. Cronin SJF, Nehme NT, Limmer S et al (2009) Genome-wide RNAi screen identifies genes involved in intestinal pathogenic bacterial infection. Science 325:340–343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Klass MR (1983) A method for the isolation of longevity mutants in the nematode Caenorhabditis elegans and initial results. Mech Ageing Dev 22:279–286

    Article  CAS  PubMed  Google Scholar 

  21. Friedman DB, Johnson TE (1988) A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility. Genetics 118:75–86

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kenyon C, Chang J, Gensch E et al (1993) A C. elegans mutant that lives twice as long as wild type. Nature 366:461–464

    Article  CAS  PubMed  Google Scholar 

  23. Ewbank JJ (2006) Signaling in the immune response. In: The C. elegans Research Community (ed) WormBook. doi: 10.1895/wormbook.1.83.1

    Google Scholar 

  24. Sinha A, Rae R, Iatsenko I, Sommer RJ (2012) System wide analysis of the evolution of innate immunity in the nematode model species Caenorhabditis elegans and Pristionchus pacificus. PLoS One 7, e44255. doi:10.1371/journal.pone.0044255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Garsin DA, Villanueva JM, Begun J et al (2003) Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens. Science 300:1921

    Article  CAS  PubMed  Google Scholar 

  26. Sinha A, Rae R (2014) A functional genomic screen for evolutionarily conserved genes required for lifespan and immunity in germline-deficient C. elegans. PLoS One 9:e101970. doi:10.1371/journal.pone.0101970

    Article  PubMed  PubMed Central  Google Scholar 

  27. Iatsenko I, Sinha A, Rödelsperger C, Sommer RJ (2013) New role for DCR-1/Dicer in Caenorhabditis elegans innate immunity against the highly virulent bacterium Bacillus thuringiensis DB27. Infect Immun 81:3942–3957

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Grishok A, Pasquinelli AE, Conte D et al (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23–34

    Article  CAS  PubMed  Google Scholar 

  29. Min K, Kang J, Lee J (2010) A modified feeding RNAi method for simultaneous knock-down of more than one gene in Caenorhabditis elegans. Biotechniques 48:229–232

    Article  CAS  PubMed  Google Scholar 

  30. Stiernagle T (2006) Maintenance of C. elegans. In: The C. elegans Research Community (ed) WormBook. doi: 10.1895/wormbook.1.101.1

  31. Kimble JE, White JG (1981) On the control of germ cell development in Caenorhabditis elegans. Dev Biol 81:208–219

    Article  CAS  PubMed  Google Scholar 

  32. Maine EM (2001) RNAi As a tool for understanding germline development in Caenorhabditis elegans: uses and cautions. Dev Biol 239:177–189

    Article  CAS  PubMed  Google Scholar 

  33. Rae R, Riebesell M, Dinkelacker I et al (2008) Isolation of naturally associated bacteria of necromenic Pristionchus nematodes and fitness consequences. J Exp Biol 211:1927–36

    Article  CAS  PubMed  Google Scholar 

  34. Rae R, Iatsenko I, Witte H, Sommer RJ (2010) A subset of naturally isolated Bacillus strains show extreme virulence to the free-living nematodes Caenorhabditis elegans and Pristionchus pacificus. Environ Microbiol 12:3007–3021

    Article  CAS  PubMed  Google Scholar 

  35. Yang J-S, Nam H-J, Seo M et al (2011) OASIS: online application for the survival analysis of lifespan assays performed in aging research. PLoS One 6, e23525. doi:10.1371/journal.pone.0023525

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Thakur N, Pujol N, Tichit L, Ewbank JJ (2014) Clone mapper: an online suite of tools for RNAi experiments in Caenorhabditis elegans. G3 (Bethesda) 4:2137–2145

    Article  Google Scholar 

  37. Schmitz C, Kinge P, Hutter H (2007) Axon guidance genes identified in a large-scale RNAi screen using the RNAi-hypersensitive Caenorhabditis elegans strain nre-1(hd20) lin-15b(hd126). Proc Natl Acad Sci U S A 104:834–839

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Calixto A, Chelur D, Topalidou I et al (2010) Enhanced neuronal RNAi in C. elegans using SID-1. Nat Methods 7:554–559

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Firnhaber C, Hammarlund M (2013) Neuron-specific feeding RNAi in C. elegans and its use in a screen for essential genes required for GABA neuron function. PLoS Genet 9(11):e1003921. doi:10.1371/journal.pgen.1003921

    Article  PubMed  PubMed Central  Google Scholar 

  40. Lezzerini M, van de Ven K, Veerman M et al (2015) Specific RNA interference in Caenorhabditis elegans by ingested dsRNA expressed in Bacillus subtilis. PLoS One 10, e0124508. doi:10.1371/journal.pone.0124508

    Article  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. University of Massachusetts Medical School, LRB 770R, 364 Plantation Street, Worcester, MA, 01605, USA

    Amit Sinha

  2. School of Natural Sciences and Psychology, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool, L3 3AF, UK

    Robbie Rae

Authors
  1. Amit Sinha
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  2. Robbie Rae
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Corresponding author

Correspondence to Robbie Rae .

Editor information

Editors and Affiliations

  1. Institute of Molecular Medicine, Phoenix Children’s Hospital, Phoenix, Arizona, USA

    David O. Azorsa

  2. Constellation Pharmaceuticals, Cambridge, Massachusetts, USA

    Shilpi Arora

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Sinha, A., Rae, R. (2016). Genome-Wide RNAi Screens in C. elegans to Identify Genes Influencing Lifespan and Innate Immunity. In: Azorsa, D., Arora, S. (eds) High-Throughput RNAi Screening. Methods in Molecular Biology, vol 1470. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6337-9_14

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  • DOI: https://doi.org/10.1007/978-1-4939-6337-9_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6335-5

  • Online ISBN: 978-1-4939-6337-9

  • eBook Packages: Springer Protocols

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