RNA Interference in Cultured Drosophila Cells

Carolyn A. Worby1, Jack E. Dixon1

1 University of California at San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 26.5
DOI:  10.1002/0471142727.mb2605s65
Online Posting Date:  February, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

RNA interference (RNAi) can be used to silence genes in a number of species, including plants, nematodes, protozoans, Drosophila melanogaster, mouse embryos, and mammalian and Drosophila cell cultures. Drosophila cell culture provides the opportunity to study signal transduction pathways and protein function in a simple, well defined cell culture paradigm. Furthermore, because Drosophila are RNAi responsive, the results obtained from experiments performed on cultured cells can be confirmed in the whole organism. RNAi takes advantage of the unique ability of double‐stranded RNA (dsRNA) molecules to induce gene silencing in a highly specific manner. This phenomenon is efficacious and long lived, being passed to subsequent generations in Drosophila cell culture. To date, many Drosophila cell lines tested respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested have been efficacious at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss of function studies applicable to a wide variety of questions.

Keywords: RNA interference; Drosophila cell culture; double‐stranded RNAs

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Treatment of Drosophila Cells with dsRNA
  • Support Protocol 1: Preparation of PCR Templates
  • Support Protocol 2: Preparation of Double‐Stranded RNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Treatment of Drosophila Cells with dsRNA

  Materials
  • Drosophila cells (e.g., S2 cells; Table 26.5.1)
  • S2 growth medium (see recipe)
  • Schneider's Drosophila medium (Life Technologies)
  • dsRNA (see protocol 3)
  • 6‐well tissue culture dishes
  • Additional reagents and equipment for counting cells ( appendix 3F), assaying RNA depletion—i.e., Northern blot (unit 4.9), RNase protection assay (unit 4.7), or RT‐PCR (unit 15.5)—and immunoblotting (unit 10.8)

Support Protocol 1: Preparation of PCR Templates

  Materials
  • cDNA encoding the protein targeted for ablation (see Critical Parameters)
  • DNA polymerase, 1× buffer, and Mg2+ stock (if required): Applied Biosystems AmpliTaq Gold (unit 3.5) or NEB Vent DNA polymerase
  • 10 mM (each) dNTPs (dATP, dCTP, dGTP, dTTP; unit 15.1)
  • High Pure PCR Purification kit (Roche Molecular Biochemicals)
  • DEPC‐treated H 2O (unit 4.1)
  • Additional reagents and equipment for purification of DNA (units 2.1& 2.1; optional), and agarose gel electrophoresis and ethidium bromide staining (unit 2.5)

Support Protocol 2: Preparation of Double‐Stranded RNA

  Materials
  • MEGAscript T7 kit (Ambion):
    • T7 enzyme mix
    • 10× T7 reaction buffer
    • 75 mM ATP, CTP, GTP, and UTP solutions
    • Nuclease‐free H 2O
  • DNA, purified (see protocol 2)
  • DEPC‐treated H 2O (unit 4.1)
  • 3 M ammonium acetate, pH 4.0
  • 100% ethanol
  • Additional reagents and equipment for determining nucleic acid concentration ( appendix 3D) and visualization of dsRNA by agarose gel electrophoresis and ethidium bromide staining (unit 2.5)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Alvarado, A.S. and Newmark, P.A. 1999. Double‐stranded RNA specifically disrupts gene expression during planarian regeneration. Proc. Natl. Acad. Sci. U.S.A. 96:5049‐5054.
   Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363‐366.
   Caplen, N.J., Parrish, S., Imani, F., Fire, A., and Morgan, R.A. 2001. Specific inhibition of gene expression by small double‐stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. U.S.A. 98:9742‐9747.
   Clemens, J.C., Worby, C.A., Simonson‐Leff, N., Muda, M., Maehama, T., Hemmings, B.A., and Dixon, J.E. 2000. Use of double‐stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. U.S.A. 97:6499‐6503.
   Echalier, G. and Ohanessian, A. 1969. Isolation, in tissue culture, of Drosophila melanogaster cell lines. C.R. Acad. Sci. Hebd. Seances Acad. Sci. D 268:1771‐1773.
   Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001a. Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494‐498.
   Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. 2001b. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877‐6888.
   Fire, A., Xu, S.Q., 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.
   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.
   Ketting, R.F., Fischer, S.E.J., Bernstein, E., Sijen, T., Hannon, G.J., and Plasterk, R.H.A. 2001. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15:2654‐2659.
   Knight, S.W. and Bass, B.L. 2001. A role for the RNase III enzyme DCR‐1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293:2269‐2271.
   Misquitta, L. and Paterson, B.M. 1999. Targeted disruption of gene function in Drosophila by RNA interference (RNA‐i): A role for nautilus in embryonic somatic muscle formation. Proc. Natl. Acad. Sci. U.S.A. 96:1451‐1456.
   Montgomery, M.K. and Fire, A. 1998. Double‐stranded RNA as a mediator in sequence‐specific genetic silencing and co‐suppression. Trends Genet. 14:255‐258.
   Ngo, H., Tschudi, C., Gull, K., and Ullu, E. 1998. Double‐stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc. Natl. Acad. Sci. U.S.A. 95:14687‐14692.
   Peel, D.J., Johnson, S.A., and Milner, M.J. 1990. The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines. Tissue Cell. 22:749‐758.
   Schneider, I. 1972. Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27:353‐65.
   Tuschl, T., Zamore, P.D., Lehmann, R., Bartel, D.P., and Sharp, P.A. 1999. Targeted mRNA degradation by double‐stranded RNA in vitro. Genes Dev. 13:3191‐3197.
   Ui‐Tei, K., Nishihara, S., Sakuma, M., Togashi, S., Ueda, R., Miyata, Y., and Miyake, T. 1994. Newly established cell lines from Drosophila larval CNS express neural specific characteristics. In Vitro Cell Dev. Biol. Anim. 30A:209‐216.
   Yanagawa, S., Lee, J.S., and Ishimoto, A. 1998. Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling. J. Biol. Chem. 273:32353‐32359.
   Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. 2000. RNAi: Double‐stranded RNA directs the ATP‐dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25‐33.
Key Reference
   Clemens, et al., 2000. See above.
  A concise description of performing RNAi experiments in Drosophila tissue culture cells is contained in this reference.
Internet Resources
   http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/95/pl1?ijkey=d2gOuU9T1u.7k&keytype=ref&siteid=sigtrans
  This site provides experimental protocols for using dsRNA in Drosophila cell culture; however, the user must have a subscription to Science magazine to access it.
   http://flyrnai.org
  This site provides a description of the Drosophila RNAi Screening Center (DRSC), a facility specializing in genome wide RNAi screening. Protocols for using dsRNA in Drosophila cell culture and results from RNAi screens can also be found at this site.
   http://www.fruitfly.org
  The Berkeley Drosophila genome project (BDGP) is a consortium of the Drosophila Genome Center, funded by the National Human Genome Research Institute, National Cancer Institute, and Howard Hughes Medical Institute, supporting the work in the Gerald Rubin and Allan Spradling laboratories. Drosophila expressed sequence tags (ESTs), can be obtained from this site.
   http://www.openbiosystems.com/drosophila_rnai_collection.php
  Drosophila ESTs can be obtained from this company. In addition, PCR templates containing T7 promoter sequences at the 5′‐ends are purchasable.
   http://www.ambion.com
  Informative Web site for RNAi experiments.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library