High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei

• Published in: Nature protocols Vol. 1; no. 1; pp. 346 - 356
• Main Authors: Janse, Chris J, Ramesar, Jai, Waters, Andrew P
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01-06-2006
• Subjects: Animals; Antimalarials - pharmacology; Antiparasitic agents; Cultivation; Culture Techniques; Deoxyribonucleic acid; Dihydrofolate reductase; DNA; Drug Resistance; Efficiency; Genes; Genetic aspects; Genetic engineering; Genetic Markers; Genetic transformation; Genomes; Health aspects; Humans; Injection; Laboratory animals; Malaria; Mice; Mutation; Parasites; Physiological aspects; Plasmodium; Plasmodium berghei; Plasmodium berghei - drug effects; Plasmodium berghei - genetics; Plasmodium berghei - growth & development; Protocol; Pyrimethamine - pharmacology; Rodents; Tetrahydrofolate Dehydrogenase - genetics; Toxoplasma gondii; Transfection; Transfection - methods; Transformation, Genetic; Triazines - pharmacology; Vector-borne diseases; Netherlands
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2006.53

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.