Stable transfection of malaria parasite blood stages.
Genetic manipulation of malaria parasites would revolutionize the study of this group of pathogens and have implications for vaccine and drug development. This report describes the stable, drug-selectable genetic transformation of the clinically relevant intracellular blood stages of a malaria parasite. A plasmid transfection vector carrying the gene locus that encodes a drug-resistant form of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from the rodent malaria parasite Plasmodium berghei was constructed. Derivatives of this vector were introduced into merozoites of P. berghei by electroporation, and parasites were selected for successful transformation in the rodent host on the basis of resistance to pyrimethamine. The plasmids were present in a circular, unrearranged form that replicated episomally to an observed maximum of 15 copies per cell in drug-resistant populations.
van Dijk
M R
MR
Department of Parasitology, University of Leiden, Netherlands.
Waters
A P
AP
Janse
C J
CJ
D000818:Animals; D001483:Base Sequence; D004261:DNA Replication; D004351:Drug Resistance; D018274:Electroporation; D004912:Erythrocytes / Q000469:parasitology; D017125:Genes, Protozoan; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D010957:Plasmids; D010962:Plasmodium berghei / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D017354:Point Mutation; D011739:Pyrimethamine / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D018741:Replication Origin; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*; D014162:Transfection
D009097:Multienzyme Complexes; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D011739:Pyrimethamine