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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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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

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  1. Chris J Janse
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  2. Jai Ramesar
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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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