摘要
Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about ∼30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7–14 days) and that it involves homologous recombination favored by presence of the same P. falciparum5′hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.
摘要译文
虽然已知重组对于在人类疟疾寄生虫恶性疟原虫中产生多样性是重要的，但转染效率低以及仅在长时间（通常12周或更长时间）后才能观察到转染的DNA整合到染色体中的事实难以遗传操纵这种主要人类病原体的血液阶段。在这里，我们显示恶性疟原虫系与两种质粒的共转染，一种表达绿色荧光蛋白（gfp）报告基因，另一种表达抗药性标记（Tgdhfr-ts M23），允许选择其中的一个群体。 ~30％的寄生虫产生GFP。在这些产生GFP的寄生虫中，转染的质粒已重组成大至20kb的嵌合附加体，并且可在药物压力下维持至少16周。我们的数据表明嵌合体形成早期发生（检测到7-14天）并且它涉及同源重组，有利于存在相同的恶性疟原虫5'hrp3 UTR促进每个质粒的转录。这表明血液阶段寄生虫中存在高水平的同源重组活性，其可用于驱动新引入的DNA的快速重组，重组的研究机制，并引入用于恶性疟原虫中反式表达的基因。
MadhusudanKadekoppala[a][b];PaulCheresh[a][b];DrewCatron[a][b];Dar-derJi[a][b];KirkDeitsch[c];Thomas E.Wellems[c];H.S.Seifert[b];KasturiHaldar[a][b];. Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum[J]. Molecular and Biochemical Parasitology, 2001,112(2): 211-218