Nkrumah Louis J, Muhle Rebecca A, Moura Pedro A, Ghosh Pallavi, Hatfull Graham F, Jacobs William R, Fidock David A
Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Nat Methods. 2006 Aug;3(8):615-21. doi: 10.1038/nmeth904.
Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P. falciparum lines with the attB site integrated into the glutaredoxin-like cg6 gene. Transfection of these attB(+) lines with a dual-plasmid system, expressing a transgene on an attP-containing plasmid together with a drug resistance gene and the integrase on a separate plasmid, produced recombinant parasites within 2 to 4 weeks that were genetically uniform for single-copy plasmid integration. Integrase-mediated recombination resulted in proper targeting of parasite proteins to intra-erythrocytic compartments, including the apicoplast, a plastid-like organelle. Recombinant attB x attP parasites were genetically stable in the absence of drug and were phenotypically homogeneous. This system can be exploited for rapid genetic integration and complementation analyses at any stage of the P. falciparum life cycle, and it illustrates the utility of Bxb1-based integrative recombination for genetic studies of intracellular eukaryotic organisms.
在此，我们报道了一种高效、位点特异性的基因整合系统，可将其整合到恶性疟原虫的染色体中。这一过程由分枝杆菌噬菌体Bxb1整合酶介导，该酶催化导入的attP位点与染色体attB位点之间的重组。我们构建了attB位点整合到类谷氧还蛋白cg6基因中的恶性疟原虫株系。用双质粒系统转染这些attB(+)株系，该双质粒系统在含attP的质粒上表达一个转基因，同时在另一个单独的质粒上表达一个耐药基因和整合酶，在2至4周内产生了重组寄生虫，这些寄生虫对于单拷贝质粒整合而言在基因上是一致的。整合酶介导的重组导致寄生虫蛋白正确靶向红细胞内区室，包括顶质体，一种类似质体的细胞器。重组的attB×attP寄生虫在无药物的情况下基因稳定，并且表型均一。该系统可用于在恶性疟原虫生命周期的任何阶段进行快速基因整合和互补分析，并且它说明了基于Bxb1的整合重组在细胞内真核生物基因研究中的实用性。
