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Highly efficient subcloning of rodent malaria parasites by injection of single merosomes or detached cells

Nature Protocols volume 4, pages 1433–1439 (2009)Cite this article

Abstract

This protocol describes a method for obtaining rodent Plasmodium parasite clones with high efficiency, which takes advantage of the normal course of Plasmodium in vitro exoerythrocytic development. At the completion of development, detached cells/merosomes form, which contain hundreds to thousands of merozoites. As all parasites within a single detached cell/merosome derive from the same sporozoite, we predicted them to be genetically identical. To prove this, hepatoma cells were infected simultaneously with a mixture of Plasmodium berghei sporozoites expressing either GFP or mCherry. Subsequently, individual detached cells/merosomes from this mixed population were selected and injected into mice, resulting in clonal blood stage parasite infections. Importantly, as a large majority of mice become successfully infected using this protocol, significantly less mice are necessary than for the widely used technique of limiting dilution cloning. To produce a clonal P. berghei blood stage infection from a non-clonal infection using this procedure requires between 4 and 5 weeks.

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Figure 1: Collection of individual detached cells/merosomes under the microscope.
Figure 2: Analysis of blood stage parasites resulting from the injection of individual detached cells/merosomes.

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Acknowledgements

We thank Chris Janse and Andrew Waters for supplying GFPCON parasites.

Author information

Author notes

  1. Rebecca R Stanway and Stefanie Graewe: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

    Rebecca R Stanway, Stefanie Graewe, Annika Rennenberg, Susanne Helm & Volker T Heussler

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  1. Rebecca R Stanway
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  2. Stefanie Graewe
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  3. Annika Rennenberg
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  4. Susanne Helm
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  5. Volker T Heussler
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Contributions

R.R.S. and V.T.H. conceived the method. A.R., S.G., S.H. and R.R.S. carried out the experiments. R.R.S., S.G. and V.T.H. prepared the paper. All authors corrected the paper.

Corresponding author

Correspondence to Rebecca R Stanway.

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Stanway, R., Graewe, S., Rennenberg, A. et al. Highly efficient subcloning of rodent malaria parasites by injection of single merosomes or detached cells. Nat Protoc 4, 1433–1439 (2009). https://doi.org/10.1038/nprot.2009.172

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