Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine.

• DOI: 10.1073/pnas.93.3.1130
• Publication Date: 2002-07-26T10:34:16Z
• Authors: Y Wu; L A Kirkman; T E Wellems
• Keywords: DNA Replication; Base Sequence; Recombinant Fusion Proteins; Molecular Sequence Data; Plasmodium falciparum; Drug Resistance; Chromosome Mapping; Thymidylate Synthase; Transfection; Polymerase Chain Reaction; 3. Good health; Antimalarials; Tetrahydrofolate Dehydrogenase; 03 medical and health sciences; Pyrimethamine; Transformation, Genetic; 0302 clinical medicine; Animals; Folic Acid Antagonists; Toxoplasma; DNA Primers; Plasmids

Plasmodium falciparum malaria parasites were transformed with plasmids containing P. falciparum or Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (dhfr-ts) coding sequences that confer resistance to pyrimethamine. Under pyrimethamine pressure, transformed parasites were obtained that maintained the transfected plasmids as unrearranged episomes for several weeks. These parasite populations were replaced after 2 to 3 months by parasites that had incorporated the transfected DNA into nuclear chromosomes. Depending upon the particular construct used for transformation, homologous integration was detected in the P. falciparum dhfr-ts locus (chromosome 4) or in hrp3 and hrp2 sequences that were used in the plasmid constructs as gene control regions (chromosomes 13 and 8, respectively). Transformation by homologous integration sets the stage for targeted gene alterations and knock-outs that will advance understanding of P. falciparum.