Skip to main content

Transfection of the African and American Trypanosomes

  • Protocol

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 47))

Abstract

The protozoan genus Trypanosoma contains several pathogenic parasites of major medical and veterinary importance. These include Trypanosoma brucei subspecies, the causative agents of African trypanosomiasis, and Trypanosoma cruzi, the causative agent of American trypanosomiasis or Chagas’ disease. The development of transformation procedures for these organisms has been a major technical advance. Applications include investigation of the molecular determinants of virulence, functional analysis of potential chemotherapeutic targets, and studies on the mechanisms of gene expression, drug resistance, and developmental regulation. Electroporation is the predominant method used to transfect trypanosomes. The protocols described here are in routine use in our respective laboratories in Amsterdam (T. brucei) and London (T. cruzi), and have proven to be straightforward and reliable.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. ten Asbroek, A. L. M. A., Ouellette, M., and Borst, P. (1990) Targeted insertion of the neomycin phosphotransferase gene into the tubulin gene cluster of Trypanosoma brucei. Nature 348, 174,175.

    Article  PubMed  Google Scholar 

  2. Lee, M. G.-S. and Van der Ploeg, L. H. T. (1990) Homologous recombination and stable transfection in the parasitic protozoan Trypanosoma brucei. Science 250, 1583–1587.

    Article  PubMed  CAS  Google Scholar 

  3. Eid, J., and Sollner-Webb, B. (1991) Stable, integrative transformation of Trypanosoma brucei that occurs exclusively by homologous recombination. Proc. Natl. Acad. Sci. USA 88, 864–868.

    Article  Google Scholar 

  4. ten Asbroek, A. L. M A., Mol, C. A. A. M., Kieft, R., and Borst, P. (1993) Stable transformation of Trypanosoma brucei. Mol. Biochem. Parasit. 59, 133–142.

    Article  Google Scholar 

  5. Zomerdijk, J. C. B. M., Kieft, R., Shiels, P. G., and Borst, P. (1991) Alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a VSG gene expression site and for the ribosomal RNA genes. Nucleic Acids Res. 19, 5153–5158.

    Article  PubMed  CAS  Google Scholar 

  6. Zomerdijk, J. C. B. M., Oullette, M., ten Asbroek, A. L. M. A., Kieft, R., Bommer, A. M. M., Clayton, C. E., and Borst P. (1990) The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei. EMBO J. 9, 2791–2801.

    PubMed  CAS  Google Scholar 

  7. Jefferies, D., Tebabi, P., and Pays, E (1991) Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level. Mol. Cell. Biol. 11, 338–343.

    PubMed  CAS  Google Scholar 

  8. Rudenko, G., Le Blancq, S., Smith, J., Lee, M. G.-S., Rattray, A., and Van der Ploeg, L. H. T. (1990) Procyclic acidic repetitive protein (PARP) genes located in an unusually small α-amanitin resistant transcription unit: PARP promoter activity assayed by transient DNA transfection of Trypanosoma brucei. Mol. Cell. Biol. 10, 3492–3504.

    PubMed  CAS  Google Scholar 

  9. Sherman, D. R., Janz, L., Hug, M., and Clayton, C. (1991) Anatomy of the parp gene promoter of Trypanosoma brucei. EMBO J. 10, 3379–3386.

    PubMed  CAS  Google Scholar 

  10. Brown, S. D., Huang, J., and Van der Ploeg, L. H. T. (1992) The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters. Mol. Cell. Biol. 12, 2644–2652.

    PubMed  CAS  Google Scholar 

  11. Carruthers, V. and Cross, G. A. M. (1992) DNA-mediated transformation of bloodstream-form Trypanosoma brucei. Nucleic Acids Res. 21, 2537,2538.

    Article  Google Scholar 

  12. Otsu, K., Donelson, J. E., and Kirchhoff, L. V. (1993) Interruption of a Trypanosoma cruzi gene encoding a protein encoding 14-amino acid repeats by targeted insertion of the neomycin phosphotransferase gene. Mol. Biochem. Parasitol. 57, 317–330.

    Article  PubMed  CAS  Google Scholar 

  13. Hariharan, S., Ajioka, J., and Swindle, J. (1993) Stable transformation of Trypanosoma cruzi: inactivation of the PUB 12.5 polyubiquitin gene by targeted gene disruption. Mol. Biochem. Parasitol. 57, 15–30.

    Article  PubMed  CAS  Google Scholar 

  14. Kelly, J. M., Ward, H. M., Miles, M. A., and Kendall, G. (1992) A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania. Nucleic Acids Res. 20, 3963–3969.

    Article  PubMed  CAS  Google Scholar 

  15. Kelly, J. M., Das, P., and Tomás, A. M. (1994) An approach to function complementation by introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani using a cosmid shuttle vector. Mol. Biochem. Parasit. 65, 51–62.

    Article  CAS  Google Scholar 

  16. Kendall, G., Wilderspin, A. F., Ashall, F., Miles, M. A., and Kelly, J. M. (1990) Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase does not conform to the “hotspot” topogenic signal model. EMBO J. 9, 2751–2758.

    PubMed  CAS  Google Scholar 

  17. Brun, G. A., and Schönenberger, M. (1979) Cultivation and in vitro cloning of procyclic culture forms of T. brucei in semi-defined medium. Acta. Trop. 36, 289–292.

    PubMed  CAS  Google Scholar 

  18. Hirumi, H., and Hirumi, K. (1989) Continuous cultivation of Trypanosoma brucei bloodstream-forms in a medium containing a low concentration of serum proteins without feeder layers. J. Parasit. 75, 985–989.

    Article  PubMed  CAS  Google Scholar 

  19. Brener, Z. (1984) Laboratory-acquired Chagas’ disease: an endemic disease among parasitologists? in Genes and Antigens of Parasites—A Laboratory Manual, 2nd ed. (Morel, C. M., ed.), Fundaçâo Oswaldo Cruz, Rio de Janeiro, pp. 3–10.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1995 Humana Press Inc.

About this protocol

Cite this protocol

Kelly, J.M., Taylor, M.C., Rudenko, G., Blundell, P.A. (1995). Transfection of the African and American Trypanosomes. In: Nickoloff, J.A. (eds) Electroporation Protocols for Microorganisms. Methods in Molecular Biology™, vol 47. Humana Press. https://doi.org/10.1385/0-89603-310-4:349

Download citation

  • DOI: https://doi.org/10.1385/0-89603-310-4:349

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-310-8

  • Online ISBN: 978-1-59259-534-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics