Gene targeting in the rodent malaria parasite Plasmodium yoelii

• Published in: Molecular and biochemical parasitology 2001-04, Vol.113 (2), p.271-278
• Main Authors: Mota, Maria M, Thathy, Vandana, Nussenzweig, Ruth S, Nussenzweig, Victor
• Format: Article
• Language: English
• Subjects: Animals; Antimalarials - pharmacology; DHFR gene; Gene Deletion; Gene Targeting; gfp gene; Malaria; Malaria - parasitology; Mice; Mice, Inbred BALB C; Multienzyme Complexes - drug effects; Multienzyme Complexes - genetics; Phenotype; Plasmids - genetics; Plasmodium yoelii; Plasmodium yoelii - drug effects; Plasmodium yoelii - genetics; Plasmodium yoelii - growth & development; Plasmodium yoelii - pathogenicity; Protozoan Proteins - genetics; pyrimethamine; Pyrimethamine - pharmacology; Tetrahydrofolate Dehydrogenase - drug effects; Tetrahydrofolate Dehydrogenase - genetics; thrombospondin-related anonymous protein; Thymidylate Synthase - drug effects; Thymidylate Synthase - genetics; Transfection; Transformation, Genetic; TRAP; TRAP gene; ts gene
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(01)00228-6

It is anticipated that the sequencing of Plasmodium falciparum genome will soon be completed. Rodent models of malaria infection and stable transformation systems provide powerful means of using this information to study gene function in vivo. To date, gene targeting has only been developed for one rodent malaria species, Plasmodium berghei. Another rodent species, Plasmodium yoelii, however, is favored to study the mechanisms of protective immunity to the pre-erythrocytic stages of infection and vaccine development. In addition, it offers the opportunity to investigate unique aspects of pathogenesis of blood stage infection. Here, we report on the stable transfection and gene targeting of P. yoelii. Purified late blood stage schizonts were used as targets for electroporation with a plasmid that contains a pyrimethamine-resistant form of the P. berghei dihydrofolate reductase-thymidylate synthase (Pb dhfr-ts) fused to green fluorescent protein ( gfp) gene. After drug selection, fluorescent parasites contained intact, non-rearranged plasmids that remain stable under drug-pressure. In addition, we used another dhfr-ts/ gfp based plasmid to disrupt the P. yoelii trap (thrombospondin-related anonymous protein) locus by site-specific integration. The phenotype of P. yoelii TRAP knockout was identical to that previously reported for the P. berghei TRAP knockout. In the absence of TRAP, the erythrocytic cycle, gametocyte and oocyst development of the mutant parasites were indistinguishable from wild type (WT). Although the sporozoites appeared morphologically normal, they failed to glide and to invade the salivary glands of mosquitoes.