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Analysis of Gene Function in Trypanosoma brucei Using RNA Interference

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 265)

Abstract

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. Currently, the method of choice to examine gene function in these organisms is RNA interference (RNAi). mRNA degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. In this chapter, we describe some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA and analysis of dsRNA, and target mRNA.

Key Words

DNA transfection hairpin construct Trypanosoma brucei clonal cell line RNA dot blot inducible promoter 

References

  1. 1.
    Hannon, G. J. (2002) RNA interference. Nature 418(6894), 244–251.PubMedCrossRefGoogle Scholar
  2. 2.
    Wang, Z., Morris, J. C., Drew, M. E., and Englund, P. T. (2000) Inhibition of Trypanosoma brucei gene expression by RNA interference using an integratable vector with opposing T7 promoters. J. Biol. Chem. 275, 40,174–40,179.PubMedCrossRefGoogle Scholar
  3. 3.
    Wang, Z. and Englund, P. T. (2001) RNA interference of a trypanosome topoisomerase II causes progressive loss of mitochondrial DNA. EMBO J. 20, 4674–4683.PubMedCrossRefGoogle Scholar
  4. 4.
    Bastin, P., Ellis, K., Kohl, L., and Gull, K. (2000) Flagellum ontogeny in trypanosomes studied via an inherited and regulated RNA interference system. J. Cell Sci. 113, 3321–3328.PubMedGoogle Scholar
  5. 5.
    Drozdz, M., Palazzo, S. S., Salavati, R., O’Rear, J., Clayton, C., and Stuart, K. (2002) TbMP81 is required for RNA editing in Trypanosoma brucei. EMBO J. 21, 1791–1799.PubMedCrossRefGoogle Scholar
  6. 6.
    Wirtz, E., Leal, S., Ochatt, C., and Cross, G. A. (1999) A tightly regulated inducible expression system for conditional gene knock-outs and dominantnegative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 99, 89–101.PubMedCrossRefGoogle Scholar
  7. 7.
    Cunningham, I. (1977) New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J. Protozool. 24, 325–329.PubMedGoogle Scholar
  8. 8.
    Shi, H., Djikeng, A., Mark, T., Wirtz, E., Tschudi, C., and Ullu, E. (2000) Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA. RNA 6, 1069–1076.PubMedCrossRefGoogle Scholar
  9. 9.
    LaCount, D. J., Bruse, S., Hill, K. L., and Donelson, J. E. (2000) Double-stranded RNA interference in Trypanosoma brucei using head-to-head promoters. Mol. Biochem. Parasitol. 111, 67–76.PubMedCrossRefGoogle Scholar
  10. 10.
    Carruthers, V. B. and Cross, G. A. (1992) High-efficiency clonal growth of bloodstream-and insect-form Trypanosoma brucei on agarose plates. Proc. Natl. Acad. Sci. USA 89, 8818–8821.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  1. 1.Department of Internal MedicineYale University School of MedicineNew Haven
  2. 2.Department of Epidemiology and Public HealthYale University School of MedicineNew Haven
  3. 3.Department of Cell BiologyYale University School of MedicineNew HavenCT

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