Gene targeting in the rodent malaria parasite Plasmodium yoelii

It is anticipated that the sequencing of Plasmodium falciparum genome will soon be completed. Rodent models of malaria infection and stable transforma tion 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, Plasmod ium yoelii. however, is favored to study the mechanisms of protective immun ity to the pre-erythrocytic stages of infection and Vaccine development. In addition, it offers the opportunity to investigate unique aspects of patho genesis; of blood stage infection. Here, we report on the stable transfecti on and gene targeting of P. yoelii. Purified late blood stage schizonts wer e used as targets for electroporation with a plasmid that contains a pyrime thamine-resistant form of the P. berghei dihydrofolate reductase-thymidylat e synthase (Pbdhfr-ts) fused to green fluorescent protein (gfp) gene. After drug selection, fluorescent parasites contained intact, non-rearranged pla smids that remain stable under drug-pressure. In addition, we used another dhfr-ts/gfp based plasmid to disrupt the P. yoelii trap (thrombospondin-rel ated anonymous protein) locus by site-specific integration. The phenotype o f 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 indistinguis hable from wild type (WT). Although the sporozoites appeared morphologicall y normal, they failed to glide and to invade the salivary glands of mosquit oes. (C) 2001 Elsevier Science B.V. All rights reserved.