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Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

TitleRapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.
Publication TypeJournal Article
Year of Publication2001
AuthorsKadekoppala M, Cheresh P, Catron D, Ji DD, Deitsch K, Wellems TE, Seifert HS, Haldar K
JournalMol Biochem Parasitol
Volume112
Issue2
Pagination211-8
Date Published2001 Feb
ISSN0166-6851
KeywordsAnimals, Blotting, Southern, DNA, Recombinant, Drug Resistance, Flow Cytometry, Genes, Reporter, Genetic Markers, Green Fluorescent Proteins, Luminescent Proteins, Male, Microscopy, Fluorescence, Molecular Sequence Data, Plasmids, Plasmodium falciparum, Pyrimethamine, Recombination, Genetic, Restriction Mapping, Transfection, Transformation, Genetic, Transgenes
Abstract

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 approximately 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. falciparum 5'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.

DOI10.1016/s0166-6851(00)00368-6
Alternate JournalMol Biochem Parasitol
PubMed ID11223128
Grant ListAI 39071 / AI / NIAID NIH HHS / United States
AI26670 / AI / NIAID NIH HHS / United States

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