Figure 1

Diagrammatic representation of strategy to generate transgenic parasites by homologous recombination of Pvdhfr-ts gene replacing endogenous dhfr-ts locus. (A) Generation of transgenic P. falciparum stably expressing wild-type Pv DHFR-TS. The endogenous Pfdhfr-ts locus was targeted via double homologous recombination with a plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and blasticidin-S deaminase (bsd) marker. Yeast cytosine deaminase-uracil phosphoribosyl transferase (yfcu) was used as negative selectable marker. (B) Generation of transgenic P. berghei stably expressing wild-type Pv DHFR-TS. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and fusion gene of human dhfr (hdhfr) and yeast yfcu as positive and negative selectable markers respectively. After double homologous recombination, the drug selectable markers are excised by single homologous recombination via the Pbdhfr-ts 3'UTR repeated sequence elements (blue boxes), whilst retaining the wild-type Pvdhfr-ts gene. (C), Generation of transgenic P. berghei stably expressing mutant Pv DHFR-TS SP21. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of mutant Pvdhfr-ts sp21 (Pvsp21) flanked with 5' and 3' UTR of Pbdhfr-ts which also serve as the sites for double homologous recombination. Specific probes for Southern analysis are located by bold horizontal line. E: Eco RI, H: Hind III, K: Kpn I.