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High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei

Nature Protocols volume 1, pages 346–356 (2006)Cite this article

Abstract

This protocol describes a method of genetic transformation for the rodent malaria parasite Plasmodium berghei with a high transfection efficiency of 10−3–10−4. It provides methods for: (i) in vitro cultivation and purification of the schizont stage;(ii) transfection of DNA constructs containing drug-selectable markers into schizonts using the nonviral Nucleofector technology; and (iii) injection of transfected parasites into mice and subsequent selection of mutants by drug treatment in vivo. Drug selection is described for two (antimalarial) drugs, pyrimethamine and WR92210. The drug-selectable markers currently in use are the pyrimethamine-resistant dihydrofolate reductase (dhfr) gene of Plasmodium or Toxoplasma gondii and the DHFR gene of humans that confer resistance to pyrimethamine and WR92210, respectively. This protocol enables the generation of transformed parasites within 10–15 d. Genetic modification of P. berghei is widely used to investigate gene function in Plasmodium, and this protocol for high-efficiency transformation will enable the application of large-scale functional genomics approaches.

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Figure 1: Flushing of 250-ml cell-culture flasks containing P. berghei–infected blood in complete RPMI1640 medium with a gas mixture of 5% CO2, 5% O2, 90% N2 using a 0.2-μm filter unit connected to the gas hose (arrow).
Figure 2: Images of cultured P. berghei schizonts in Giemsa-stained thin blood smears (light microscope, ×100 objective).
Figure 3: One-step Nycodenz density-gradient purification of schizont-infected erythrocytes from uninfected erythrocytes.
Figure 4: Images of Nycodenz-purified P. berghei schizonts in Giemsa-stained thin blood smears (light microscope, x100 oil-immersed objective).
Figure 5: Examples of standard analyses of the genotype of parasites selected by drug treatment after transfection.

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References

  1. Cowman, A.F. & Crabb, B.S. in Molecular Approaches to Malaria (Sherman, I.W. ed.) 50–67 (ASM Press, Washington, DC, 2005).

    Google Scholar 

  2. Carvalho, T.G. & Menard, R. Manipulating the Plasmodium genome. Curr. Issues Mol. Biol. 7, 39–55 (2005).

    CAS  PubMed  Google Scholar 

  3. Koning-Ward, T.F., Janse, C.J. & Waters, A.P. The development of genetic tools for dissecting the biology of malaria parasites. Annu. Rev. Microbiol. 54, 157–185 (2000).

    Article  Google Scholar 

  4. Janse, C.J. et al. High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol. Biochem. Parasitol. 145, 60–70 (2006).

    Article  CAS  Google Scholar 

  5. van Dijk, M.R., McConkey, G.A., Vinkenoog, R., Waters, A.P. & Janse, C.J. Mechanisms of pyrimethamine resistance in two different strains of Plasmodium berghei. Mol. Biochem. Parasitol. 68, 167–171 (1994).

    Article  CAS  Google Scholar 

  6. van Dijk, M.R., Waters, A.P. & Janse, C.J. Stable transfection of malaria parasite blood stages. Science 268, 1358–1362 (1995).

    Article  CAS  Google Scholar 

  7. Waters, A.P., Thomas, A.W., van Dijk, M.R. & Janse, C.J. Transfection of malaria parasites. Methods 13, 134–147 (1997).

    Article  CAS  Google Scholar 

  8. Menard, R. & Janse, C. Gene targeting in malaria parasites. Methods 13, 148–157 (1997).

    Article  CAS  Google Scholar 

  9. Sakamoto, H. et al. Towards systematic identification of Plasmodium essential genes by transposon shuttle mutagenesis. Nucleic Acids Res. 33, e174 (2005).

    Article  Google Scholar 

  10. Ecker, A., Moon, R., Sinden, R.E. & Billker, O. Generation of gene targeting constructs for Plasmodium berghei by a PCR-based method amenable to high throughput applications. Mol. Biochem. Parasitol. 145, 265–268 (2006).

    Article  CAS  Google Scholar 

  11. Maasho, K., Marusina, A., Reynolds, N.M., Coligan, J.E. & Borrego, F. Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system. J. Immunol. Methods 284, 133–140 (2004).

    Article  CAS  Google Scholar 

  12. Gresch, O. et al. New non-viral method for gene transfer into primary cells. Methods 33, 151–163 (2004).

    Article  CAS  Google Scholar 

  13. Leclere, P.G. et al. Effective gene delivery to adult neurons by a modified form of electroporation. J. Neurosci. Methods 142, 137–143 (2005).

    Article  CAS  Google Scholar 

  14. Jongco, A.M., Ting, L.M., Thathy, V., Mota, M.M. & Kim, K. Improved transfection and new selectable markers for the rodent malaria parasite Plasmodium yoelii. Mol. Biochem. Parasitol. 146, 242–250 (2006).

    Article  CAS  Google Scholar 

  15. Koning-Ward, T.F. et al. The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome. Mol. Biochem. Parasitol. 106, 199–212 (2000).

    Article  Google Scholar 

  16. Gilks, C.F., Jarra, W., Harvey-Wood, K., McLean, S.A. & Schetters, T. Host diet in experimental rodent malaria: a variable which can compromise experimental design and interpretation. Parasitology 98, 175–177 (1989).

    Article  CAS  Google Scholar 

  17. Deitsch, K., Driskill, C. & Wellems, T. Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes. Nucleic Acids Res. 29, 850–853 (2001).

    Article  CAS  Google Scholar 

  18. Mamoun, C.B. et al. Transfer of genes into Plasmodium falciparum by polyamidoamine dendrimers. Mol. Biochem. Parasitol. 103, 117–121 (1999).

    Article  CAS  Google Scholar 

  19. Gardiner, D.L., Skinner-Adams, T.S., Spielmann, T. & Trenholme, K.R. Malaria transfection and transfection vectors. Trends Parasitol. 19, 381–383 (2003).

    Article  Google Scholar 

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Acknowledgements

We would like to thank B. Franke-Fayard, H. Kroeze and S. Khan for their critical comments.

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Authors and Affiliations

  1. Leiden University Medical Center, P.O. Box 9600, Leiden, 2300, RC, The Netherlands

    Chris J Janse, Jai Ramesar & Andrew P Waters

Authors
  1. Chris J Janse
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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Purification of cultured, mature schizonts of P. berghei by Nycodenz density-gradient centrifugation. (MOV 2727 kb)

Supplementary Video 2

Transfection of schizonts of P. berghei using the Amaxa Nucleofector device and injection of transfected schizonts into a mouse. (MOV 3048 kb)

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Janse, C., Ramesar, J. & Waters, A. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat Protoc 1, 346–356 (2006). https://doi.org/10.1038/nprot.2006.53

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