Drosophila pp 139-153 | Cite as

RNAi Screening in Cultured Drosophila Cells

  • Sandra Steinbrink
  • Michael Boutros
Part of the Methods in Molecular Biology book series (MIMB, volume 420)

Abstract

RNA interference (RNAi) has become a widely used tool to analyze biological functions in vivo and in vitro. With the availability of an increasing number of Drosophila cell lines, a variety of different processes can be studied ranging from cell cycle control defects to signaling pathway activities and changes in cell morphology. Owing to the ease of RNAi in Drosophila cells, this experimental system has become a preferred method to screen for novel cellular factors, before their in depth analysis. We here describe the experimental procedures for RNAi experiments in cultured Drosophila cells, starting from the design of long double-stranded RNAs, their synthesis by in vitro transcription and application for cell-based RNAi experiments from low to high-throughput formats. Finally, we show how phenotype analysis can be performed using cell-based assays for luminescence or flow cytometric analysis as examples.

Key Words

Drosophila cells high-throughput screening phenotypic readouts RNAi double-stranded RNA cell-based assays 

References

  1. 1.
    Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.CrossRefPubMedGoogle Scholar
  2. 2.
    Kennerdell, J. R. and Carthew, R. W. (1998) Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017–1026.CrossRefPubMedGoogle Scholar
  3. 3.
    Clemens, J. C., Worby, C. A., Simonson-Leff, N., et al. (2000) Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97, 6499–6503.CrossRefPubMedGoogle Scholar
  4. 4.
    Boutros, M., Kiger, A. A., Armknecht, S., et al. (2004) Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832–835.CrossRefPubMedGoogle Scholar
  5. 5.
    Kiger, A., Baum, B., Jones, S., et al. (2003) A functional genomic analysis of cell morphology using RNA interference. J. Biol. 2, 27.CrossRefPubMedGoogle Scholar
  6. 6.
    Lum, L., Yao, S., Mozer, B., et al. (2003) Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039–2045.CrossRefPubMedGoogle Scholar
  7. 7.
    Bartscherer, K., Pelte, N., Ingelfinger, D., and Boutros, M. (2006) Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125, 523–533.CrossRefPubMedGoogle Scholar
  8. 8.
    Gesellchen, V., Kuttenkeuler, D., Steckel, M., Pelte, N., and Boutros, M. (2005) An RNA interference screen identifies Inhibitor of Apoptosis Protein 2 as a regulator of innate immune signalling in Drosophila. EMBO Rep. 6, 979–984.CrossRefPubMedGoogle Scholar
  9. 9.
    Kleino, A., Valanne, S., Ulvila, J., et al. (2005) Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway. EMBO J. 24, 3423–3434.CrossRefPubMedGoogle Scholar
  10. 10.
    Foley, E. and O’Farrell, P. H. (2004) Functional dissection of an innate immune response by a genome-wide RNAi screen. PLoS Biol. 2, E203.CrossRefPubMedGoogle Scholar
  11. 11.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  12. 12.
    Kulkarni, M. M., Booker, M., Silver, S. J., et al. (2006) Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat. Methods 3, 833–838.PubMedGoogle Scholar
  13. 13.
    Ma, Y., Creanga, A., Lum, L., and Beachy, P. A. (2006) Prevalence of off-target effects in Drosophila RNA interference screens. Nature 443, 359–363.CrossRefPubMedGoogle Scholar
  14. 14.
    Arziman, Z., Horn, T., and Boutros, M. (2005) E-RNAi: a web application to design optimized RNAi constructs. Nucleic Acids Res. 33, W582–W588.CrossRefPubMedGoogle Scholar
  15. 15.
    Rozen, S. and Skaletsky, H. (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386.PubMedGoogle Scholar
  16. 16.
    Horn, T., Arziman, Z., Berger, J., and Boutros, M. (2007) GenomeRNAi: a database for cell-based RNAi phenotypes. Nucleic Acids Res. 35, D492–D497.CrossRefPubMedGoogle Scholar
  17. 17.
    Sims, D., Bursteinas, B., Gao, Q., Zvelebil, M., and Baum, B. (2006) FLIGHT: database and tools for the integration and cross-correlation of large-scale RNAi phenotypic datasets. Nucleic Acids Res. 34, D479–D483.CrossRefPubMedGoogle Scholar
  18. 18.
    Hild, M., Beckmann, B., Haas, S., et al. (2003) An integrated gene annotation and transcriptional profiling approach towards the full gene content of the Drosophila genome. Genome Biol. 5, R3.CrossRefPubMedGoogle Scholar
  19. 19.
    Boutros, M., Bras, L., and Huber, W. (2006) Analysis of cell-based RNAi screens. Genome Biol. 7, R66.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2008

Authors and Affiliations

  • Sandra Steinbrink
    • 1
  • Michael Boutros
    • 1
  1. 1.German Cancer Research CenterHeidelbergGermany

Personalised recommendations