Targeted disruption of the tpx-2 gene in P. berghei. (A) The targeting construct, composed of the 5′ and 3′ regions (filled boxes) and partial open reading frame (hatched boxes) of the pbtpx-2 and the pyrimethamine-resistant variant of dhfr-ts of P. berghei (dhfr-ts/mt) as a selectable marker (gray arrow), was integrated into the wild-type (WT) PbTPx-2 locus by double-crossover homologous recombination. Recombination disrupts pbtpx-2 and creates the locus containing dhfr-ts/mt (PbTPx-2 KO locus), which confers pyrimethamine resistance to disruptants. (B) Southern blot analysis of genomic DNA samples from the wild-type parasite populations with pyrimethamine resistance (dhfr-ts/mt at the dhfr-ts locus) (TPx-2 WT), and two pbtpx-2 disruptant populations (TPx-2 KO). DNA samples were digested with Xba I, separated on 0.7% agarose gels, transferred to nylon membranes, and hybridized with a probe. The single 6.2-kb band in the TPx-2 KO populations indicates pbtpx-2 disruption, whereas the single 7.6-kb band in the TPx-2 WT populations indicates an intact pbtpx-2 locus.