Rapid recombination among transfected plasmids, chimeric episome formationand trans gene expression in Plasmodium falciparum

Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum. the low efficiencies of transfect ion and the fact that integration of transfected DNA into chromosomes is ob served only after long periods (typically 12 weeks or more) have made it di fficult to genetically manipulate the blood stages of this major human path ogen. Here we show that co-transfection of a P. falciparum line with two pl asmids. one expressing a green fluorescent protein (gfp) reporter and the o ther expressing a drug resistance marker (Tgdhfr-ts M23). allowed selection of a population in which about similar to 30% of the parasites product GFP . In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 30 kb and could be maintained under dru g pressure for at least 16 weeks. Our data suggest that chimera formation o ccurs early (detected by 7-14 days) and that it involves homologous recombi nation Favored by presence of the same P. falciparum 5'hrp3 UTR promoting t ranscription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be u sed to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum , (C) 2001 Elsevier Science B.V. All rights reserved.