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

Janse, C. J., Ramesar, J. and Waters, A. P. (2006) High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nature Protocols, 1(1), pp. 346-356. (doi: 10.1038/nprot.2006.53) (PMID:17406255)

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Waters, Professor Andy
• Authors: Janse, C. J., Ramesar, J., and Waters, A. P.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Nature Protocols
• Publisher: Nature Publishing Group
• ISSN: 1754-2189
• ISSN (Online): 1750-2799
• Published Online: 29 June 2006