Malaria pp 75-98 | Cite as

Transfection of Plasmodium falciparum

  • Melanie RugEmail author
  • Alexander G. Maier
Part of the Methods in Molecular Biology book series (MIMB, volume 923)


Genetic manipulation of Plasmodium falciparum remains very challenging, mainly due to the parasite genome’s high A/T-richness and low transfection efficiency. This chapter includes methods for generating transient and stable transfections by electroporation, allelic replacement with tagged genes, gene deletion, and the analysis of all the above.

Key words

Plasmodium falciparum Transfection Electroporation Vectors Positive selectable markers Negative selectable markers Knockout Allelic replacement 



Methods like the ones described above are based on incremental findings and observations made by many researchers and only a few of them can be accredited for their contributions. Many members of our laboratories past and present have refined the methods that we described above. We are very grateful to all their contributions, especially to those of Alan Cowman, Brendan Crabb, Tania de Koning-Ward, Jenny Thompson, Tony Triglia, Rebecca O’Donnell, Matthew O’Neill, and Ping Cannon. We thank the Red Cross for their continuous supply of Blood and human serum. Our laboratories are supported by the Australian National Health and Medical Research Council and the Australian Research Council.


  1. 1.
    Gardner MJ et al (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419:498–511PubMedCrossRefGoogle Scholar
  2. 2.
    Deitsch K et al (2001) Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes. Nucleic Acids Res 29:850–853PubMedCrossRefGoogle Scholar
  3. 3.
    Skinner-Adams TS et al (2003) Comparison of Plasmodium falciparum transfection methods. Malar J 2:19PubMedCrossRefGoogle Scholar
  4. 4.
    Wu Y et al (1995) Transfection of Plasmodium falciparum within human red blood cells. Proc Natl Acad Sci USA 92:973–977PubMedCrossRefGoogle Scholar
  5. 5.
    Crabb BS et al (2004) Transfection of the human malaria parasite Plasmodium falciparum. Methods Mol Biol 270:263–276PubMedGoogle Scholar
  6. 6.
    O’Donnell RA et al (2002) A genetic screen for improved plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium falciparum chromosomes. EMBO J 21:1231–1239PubMedCrossRefGoogle Scholar
  7. 7.
    Limenitakis J, Soldati-Favre D (2011) Functional genetics in Apicomplexa: potentials and limits. FEBS Lett 585:1579–1588PubMedCrossRefGoogle Scholar
  8. 8.
    Militello KT, Wirth DF (2003) A new reporter gene for transient transfection of Plasmodium falciparum. Parasitol Res 89:154–157PubMedCrossRefGoogle Scholar
  9. 9.
    de Koning-Ward TF et al (2000) The development of genetic tools for dissecting the biology of malaria parasites. Annu Rev Microbiol 54:157–185PubMedCrossRefGoogle Scholar
  10. 10.
    Mamoun CB et al (1999) A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum. Proc Natl Acad Sci USA 96:8716–8720PubMedCrossRefGoogle Scholar
  11. 11.
    de Koning-Ward TF et al (2001) Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum. Mol Biochem Parasitol 117:155–160PubMedCrossRefGoogle Scholar
  12. 12.
    Duraisingh MT et al (2002) Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination. Int J Parasitol 32:81–89PubMedCrossRefGoogle Scholar
  13. 13.
    Maier AG et al (2006) Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination. Mol Biochem Parasitol 150:118–121PubMedCrossRefGoogle Scholar
  14. 14.
    Waller RF et al (2000) Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway. EMBO J 19:1794–1802PubMedCrossRefGoogle Scholar
  15. 15.
    Rug M et al (2004) Correct promoter control is needed for trafficking of the ring-infected erythrocyte surface antigen to the host cytosol in transfected malaria parasites. Infect Immun 72:6095–6105PubMedCrossRefGoogle Scholar
  16. 16.
    Tonkin CJ et al (2004) Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method. Mol Biochem Parasitol 137:13–21PubMedCrossRefGoogle Scholar
  17. 17.
    Wickham ME et al (2001) Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes. EMBO J 20:5636–5649PubMedCrossRefGoogle Scholar
  18. 18.
    Knuepfer E et al (2005) Function of the Plasmodium export element can be blocked by green fluorescent protein. Mol Biochem Parasitol 142:258–262PubMedCrossRefGoogle Scholar
  19. 19.
    Doherty JP et al (1993) Escherichia coli host strains SURE and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains. Gene 124:29–35PubMedCrossRefGoogle Scholar
  20. 20.
    Crabb BS, Cowman AF (1996) Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum. Proc Natl Acad Sci USA 93:7289–7294PubMedCrossRefGoogle Scholar
  21. 21.
    Lai Z et al (1999) A shotgun optical map of the entire Plasmodium falciparum genome. Nat Genet 23:309–313PubMedCrossRefGoogle Scholar
  22. 22.
    Baldi DL et al (2000) RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum. EMBO J 19:2435–2443PubMedCrossRefGoogle Scholar
  23. 23.
    Maier AG et al (2003) Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations. Nat Med 9:87–92PubMedCrossRefGoogle Scholar
  24. 24.
    O’Neill MT et al (2011) Gene deletion from Plasmodium falciparum using FLP and Cre recombinases: implications for applied site-specific recombination. Int J Parasitol 41:117–123PubMedCrossRefGoogle Scholar
  25. 25.
    Kilby NJ et al (1993) Site-specific recombinases: tools for genome engineering. Trends Genet 9:413–421PubMedCrossRefGoogle Scholar
  26. 26.
    Frankland S et al (2007) Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface. Eukaryot Cell 6:1584–1594PubMedCrossRefGoogle Scholar
  27. 27.
    Spielmann T et al (2006) Reliable transfection of Plasmodium falciparum using non-commercial plasmid mini preparations. Int J Parasitol 36:1245–1248PubMedCrossRefGoogle Scholar
  28. 28.
    Allen RJ, Kirk K (2010) Plasmodium falciparum culture: the benefits of shaking. Mol Biochem Parasitol 169:63–65PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Research School of BiologyThe Australian National UniversityCanberraAustralia

Personalised recommendations