Complementation of Plasmodium berghei TRAP knockout parasites using human dihydrofolate reductase gene as a selectable marker

Previously we have used the Plasmodium dihydrofolate reductase thymidylate synthase (DHFR-TS) selectable marker to generate Plasmodium berghei TRAP nu ll mutant parasites. These TRAP null mutants do not glide and they showed a great reduction in their ability to infect mosquito salivary glands and th e hepatocytes of the vertebrate host. Thus far, complementation of these kn ockout parasites was not possible due to the lack of additional selectable markers. Recently, a new selectable marker, based on the human dihydrofolat e reductase (hDHFR) gene, has been developed which confers resistance to th e antifolate drug WR99210. This drug has been found to be highly active aga inst pyrimethamine-sensitive and -resistant strains of P. berghei. In this study, we have used the hDHFR gene as a second selectable marker for the co mplementation of P. berghei TRAP null mutant parasites. Restoration of the TRAP null mutant parasites to the wild-type phenotype was achieved in this study via autonomously replicating episomes bearing a wild-type copy of the TRAP gene. This is the first report of complementation of a mutant phenoty pe in malaria parasites. (C) 2001 Elsevier Science B.V. All rights reserved .