Mota M M, Thathy V, Nussenzweig R S, Nussenzweig V
Michael Heidelberger Division, Department of Pathology (MSB131), New York University Medical Center, 550 First Avenue, New York, NY 10016, USA.
Mol Biochem Parasitol. 2001 Apr 6;113(2):271-8. 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 (Pbdhfr-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.
预计恶性疟原虫基因组的测序工作很快将完成。疟疾感染的啮齿动物模型和稳定转化系统为利用这些信息在体内研究基因功能提供了有力手段。迄今为止，基因打靶仅在一种啮齿动物疟原虫物种——伯氏疟原虫中得以开发。然而，另一种啮齿动物疟原虫物种——约氏疟原虫，更有利于研究针对感染前红细胞阶段的保护性免疫机制以及疫苗开发。此外，它还提供了研究血期感染发病机制独特方面的机会。在此，我们报告约氏疟原虫的稳定转染和基因打靶情况。纯化的晚期血期裂殖体被用作电穿孔的靶标，所用质粒含有与绿色荧光蛋白（gfp）基因融合的伯氏疟原虫二氢叶酸还原酶-胸苷酸合成酶（Pbdhfr-ts）的乙胺嘧啶抗性形式。经过药物筛选后，荧光寄生虫含有完整的、未重排的质粒，这些质粒在药物压力下保持稳定。此外，我们使用另一种基于dhfr-ts/gfp的质粒，通过位点特异性整合来破坏约氏疟原虫陷阱（血小板反应蛋白相关无名蛋白）基因座。约氏疟原虫TRAP基因敲除的表型与先前报道的伯氏疟原虫TRAP基因敲除的表型相同。在缺乏TRAP的情况下，突变寄生虫的红细胞周期、配子体和卵囊发育与野生型（WT）无明显差异。尽管子孢子在形态上看起来正常，但它们无法滑行并侵入蚊子的唾液腺。
