Analyisis of the Plasmodial rhoptry protein function by gene targeting

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Invasive stage of the malaria parasite contains characteristic microorganelles at the apical end. One of the organelle is called as the rhoptries and thought to be involved in the critical invasion step into the host cells. The aim of this research proposal was to analyze the function of rhoptry proteins (140-kDa RhopHl and 100-kDa RhopHS proteins) by using gene-targeting technique. Because the gene-targeting technique in rodentplasmodium was only established in P. berghei, closely related species to P. yoelii, we evaluated; 1) the sensitivity to the anti-plasmodial drug, pyrimethamine, for the P. yoelii clone we maintain, 2) the efficacy of the gene-introduction into P. yoelii by transfecting the circular form of pMD204, which carry the resistance to pyrimethamine, into P. yoelii, 3) the efficient concentration of pyrimethamine for pMD204-transfected P. yoelii. The results suggested that the transfection into P. yoelii could be achieved with a slightly modified technique from that into P. berghei. The first version of the gene-targeting constructs were designed for P yoelii rhoptry proteins, PyRhopHl and PyRhopHS, and constructed by inserting PCR-amplified DNA fragments into the targeting vector pMD204 (pYRH1-Dis and pYR3-Dis, respectively). The attempt to transfect the linearized genetargeting constructs into P. yoelii and obtain the gene-disrupted clones under drug pressure were unsuccessful at this moment. To overcome this problem, we re-designed the gene-targeting constructs and the attempts to disrupt these gene loci is still underway.