<p>Abstract</p> <p>Background</p> <p>The prevalence of drug resistance amongst the human malaria <it>Plasmodium </it>species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new <it>Plasmodium falciparum </it>dihydrofolate reductase (<it>Pf</it>DHFR) mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development.</p> <p>Methods</p> <p>A system to identify mutations in <it>Pfdhfr </it>gene that confer antifolate drug resistance using an animal <it>Plasmodium </it>parasite model was developed. By using error-prone PCR and <it>Plasmodium </it>transfection technologies, libraries of <it>Pfdhfr </it>mutant were generated and then episomally transfected to <it>Plasmodium berghei </it>parasites, from which pyrimethamine-resistant <it>Pf</it>DHFR mutants were selected.</p> <p>Results</p> <p>The principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic <it>P. berghei</it>, in which endogenous <it>Pbdhfr </it>allele was replaced with the mutant <it>Pfdhfr<sup>S108N</sup></it>, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine.</p> <p>Conclusion</p> <p>This study demonstrated the power of the transgenic <it>P. berghei </it>system to predict drug-resistant <it>Pfdhfr </it>mutations in an <it>in vivo </it>parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.</p
