Experimental animals and parasite
For all experiments, female BALB/c mice (National Laboratory Animal Center, Mahidol University, Thailand) 4-6 weeks old and weighing 20-25 g were used for P. berghei parasite infections. The transgenic P. berghei parasite line MRA-867 expressing green fluorescent protein without drug-resistant selectable marker (PbGFP), kindly provided by Drs. Andrew Waters and Chris Janse of Malaria Research Group, Leiden University Medical Center, the Netherlands, was used in this study . All animal work was evaluated and approved by the Ethical Committee on Animal Experimentation, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand, and followed international guidelines for the use of animals in experimental studies.
Construction of P. berghei transfection plasmid
Plasmid for P. berghei transfection in this study was modified from the original plasmid pL0017 , which was kindly provided by Drs. Andrew Waters and Chris Janse (Leiden University Medical Centre, the Netherlands). The final transfection plasmid, designated pY005, contains wild type Pfdhfr-ts gene flanked with 1.0-kb each of 5' and 3' untranslated region (UTR) sequences of Pbdhfr-ts. Bam HI and Afl II restriction sites were introduced at 5' and 3' ends, respectively, of Pfdhfr domain to serve as cloning sites for the randomly-mutated Pfdhfr library.
Construction of Pfdhfr random mutant library
Pfdhfr mutant library was generated by error prone PCR . The 50 μL PCR reaction contained 1 ng of pY005 plasmid template harboring wild type Pfdhfr, 10 μM of sense primer F1 (CGGTGGATCC ATGATGGAACAAG; Bam HI site is underlined), 10 μM of antisense primer R1 (CTTTGTCATCATTCTTAAG AGGC; Afl II site is underlined), 0.1 mM dGTP, 0.1 mM dATP, 0.5 mM dCTP, 0.5 mM dTTP, 1× Mutagenesis buffer  and 5 units of GoTaq® DNA polymerase (Promega). The thermocycle condition was: 1 cycle of 95°C for 3 min, 30 cycles of 95°C for 1 min, 50°C for 1 min, 72°C for 1 min, and final extension of 72°C for 5 min. PCR products of random mutant Pfdhfr library of about 0.7 kb were cloned into the Bam HI/Afl II sites of pY005. The plasmids containing Pfdhfr mutation libraries were grown in Luria Bertani broth containing 100 μg/ml ampicillin in a 37°C incubator shaker for 12-16 hours. Plasmids were extracted and purified using a Qiaprep Spin Miniprep kit (Qiagen). Extracted plasmids were precipitated by isopropanol and re-suspended in 10 μl TE buffer (10 mM Tris, 1 mM EDTA pH 8.0) for use in transfection experiment.
Determination of pyrimethamine sensitivity to parental and transgenic parasites
The sensitivity of pyrimethamine to inhibit the parental PbGFP parasite or transgenic parasites expressing Pf DHFR mutant was determined by the 4-day suppressive test . Five groups of five BALB/c mice per group were infected intravenously (i.v.) with 1 × 107 parasitized erythrocytes and treated with different concentration of pyrimethamine by intraperitoneal (i.p.) injection four hours post infection. The control group was treated with 5% (v/v) DMSO in PBS pH 4.0. The experimental groups were treated with different doses of pyrimethamine through the same route for another 3 days. Twenty-four hours after the last treatment (day 4), percentages of parasitaemia were determined by microscopic counting of Giemsa-stained smears from mouse tail blood.
All statistical analyses were carried out using SigmaPlot software version 11 (Systat Software Inc., USA). For calculation of the growth inhibitory curve, parasitaemia of the control group was set as 0% inhibition. The non-linear regression for sigmoidal dose-response (variable slope) was used to calculate the 50%, 90% or 95% effective dose (ED50, ED90 or ED95) values.
Transfection, selection and identification of Pfdhfr random mutant libraries
In vitro culture of PbGFP and P. berghei transfection were performed as described . Briefly, parasitized blood was collected from a donor animal and cultured overnight in culture media (RPMI 1640 medium containing 20% heat inactivated fetal calf serum, 50 IU/mL neomycin and 25 mM Hepes). Schizont stage parasites were purified from the overnight culture by Nycodenz gradient centrifugation. The merozoites were transfected with the circular plasmid DNA harboring Pfdhfr mutant libraries using the standard Amaxa Nucleofector protocol  and re-infected into animals by i.v. injection. Twenty-four hours after transfection, 0.25 mg/kg of pyrimethamine was used to treat the infected mice by i.p. injection daily. Smears were taken to check parasitaemia and when positive, left to multiply, until parasite numbers were adequate (about 3% parasitaemia) for genomic analysis. Tail blood was drawn from infected animals on alternate days until parasitaemia reached 8-10% and genomic DNA was extracted from whole blood using a genomic DNA Mini Kit (Geneaid). The genomic DNA obtained was transformed into E. coli DH5α to recover the circular plasmid DNA containing Pfdhfr mutants. The transformed bacterial colonies were picked and extracted for plasmid DNA using a Qiaprep Spin Miniprep kit (Qiagen). The sequences of Pfdhfr mutants were obtained by DNA sequencing (Biodesign Sequencing Service, Thailand).
Transfection, selection and cloning of transgenic P. berghei parasite
Plasmid containing resistant mutant PfdhfrS108N was completely digested with Hind III and Kas I restriction enzymes to generate linear plasmid. The 5' and 3'UTRs of Pbdhfr-ts served as homologous recombination sites for replacement of the endogenous Pbdhfr-ts on chromosome 7 with PfdhfrS108N. In vitro culture of PbGFP and P. berghei transfection were performed as described . Twenty-four hours after transfection, 0.25 mg/kg of pyrimethamine was used to treat the infected mice by i.p. injection daily until the drug resistant parasites appeared. The integrated transgenic mutant parasite clones were obtained by the limiting dilution method .
PCR analysis of transgenic P. berghei parasite
The correct integration of PfdhfrS108N sequence into the genome via the 5' and 3'UTRs of the Pbdhfr-ts locus was determined by PCR. A 4.0-kb DNA fragment spanning the endogenous 5'UTR Pbdhfr-ts sequence and the introduced Pfdhfr was indicative of an integration event and was amplified using primers F2 (TTGAGCTACATAACTTCCATACAT) and R1 (described above). A 3.0-kb DNA fragment spanning the introduced Pfdhfr and the endogenous 3'UTR Pbdhfr-ts sequence, indicative of a 3' integration event was amplified using primers F1 (described above) and R2 (CGATCTACACCTCTTCAT).
Expression profile of transgenic mutant parasite
The transgenic parasites expressed Pf DHFRS108N-TS under the control of 5' and 3'UTRs of Pbdhfr-ts. To determine whether this promoter efficiently drives PfdhfrS108N-ts mRNA expression, reverse-transcription PCR (RT-PCR) was performed. Total RNA was isolated from blood stage transgenic P. berghei parasites using Trizol reagent (Invitrogen). cDNA was generated and used as template for amplification with gene specific primers for the Pfdhfr transgene using primers F1 and R1 (described above). Controls included specific primers for Pbdhfr gene: PbDTF (GGGGGGGGCATATGGAAGACTTATCTGAAACATTCG) and PbDTR (GGACTAGTGTACTTCCTCATTTGG) and P. berghei alpha tubulin gene: PbatubulinF (GCATGCTGGGAGCTATTTTG) and PbatubulinR (GCTGGTTCAAATGCTGAGTTTG). RT-PCR was performed using the same PCR condition as described above.