title: High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.
abstract: This protocol describes a method of genetic transformation for the rodent malaria parasite Plasmodium berghei with a high transfection efficiency of 10(-3)-10(-4). It provides methods for: (i) in vitro cultivation and purification of the schizont stage;(ii) transfection of DNA constructs containing drug-selectable markers into schizonts using the nonviral Nucleofector technology; and (iii) injection of transfected parasites into mice and subsequent selection of mutants by drug treatment in vivo. Drug selection is described for two (antimalarial) drugs, pyrimethamine and WR92210. The drug-selectable markers currently in use are the pyrimethamine-resistant dihydrofolate reductase (dhfr) gene of Plasmodium or Toxoplasma gondii and the DHFR gene of humans that confer resistance to pyrimethamine and WR92210, respectively. This protocol enables the generation of transformed parasites within 10-15 d. Genetic modification of P. berghei is widely used to investigate gene function in Plasmodium, and this protocol for high-efficiency transformation will enable the application of large-scale functional genomics approaches.
authors.affiliation: Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. c.j.janse@lumc.nl
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Janse
authors.forename: Jai
authors.initials: J
authors.lastname: Ramesar
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Waters
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D046508:Culture Techniques; D004351:Drug Resistance; D005819:Genetic Markers; D006801:Humans; D051379:Mice; D009154:Mutation; D010962:Plasmodium berghei / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D011739:Pyrimethamine / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014170:Transformation, Genetic; D014227:Triazines / Q000494:pharmacology
chemical_list: D000962:Antimalarials; D005819:Genetic Markers; D014227:Triazines; C006201:BRL 6231; D013762:Tetrahydrofolate Dehydrogenase; D011739:Pyrimethamine