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Nat Protoc. 2009;4(10):1433-9. doi: 10.1038/nprot.2009.172. Epub 2009 Sep 10.

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

Author information

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

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.

PMID:
19745825
DOI:
10.1038/nprot.2009.172
[Indexed for MEDLINE]

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