title: Highly efficient subcloning of rodent malaria parasites by injection of single merosomes or detached cells.
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.
authors.affiliation: Bernhard Nocht Institute for Tropical Medicine, Department of Molecular Parasitology, Hamburg, Germany.
authors.forename: Rebecca R
authors.initials: RR
authors.lastname: Stanway
authors.forename: Stefanie
authors.initials: S
authors.lastname: Graewe
authors.forename: Annika
authors.initials: A
authors.lastname: Rennenberg
authors.forename: Susanne
authors.initials: S
authors.lastname: Helm
authors.forename: Volker T
authors.initials: VT
authors.lastname: Heussler
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D006801:Humans; D007267:Injections; D008288:Malaria / Q000469:parasitology*; D051379:Mice; D010274:Parasitology / Q000379:methods*; D010962:Plasmodium berghei / Q000254:growth & development / Q000302:isolation & purification*