Figure 5

pfk1 and pfk2 knockout transfections. A. Schematic representation of the knockout transfection plasmid (pminiBSD/pfk KO), pfk loci within the parasite genome, and the resultant pfk loci after successful integration. Relative locations of primers used in the PCR detection of integration are shown. Integration-specific PCR products of pfk1 (B.) and pfk2 (C.) are shown. Expected sizes of PCR products resulting from each primer pair are shown in the table below. A ~1.0 kb product (*) was detected in d1, indicative of integration into pfk1 (panel B), whereas 1.0 kb (d1) and 1.3 kb (d2) products indicative of integration into pfk2 (panel C) were detected. Integration into the genes hrp2 and cam was also assessed, as regulatory elements of these Plasmodium sp. genes drive expression of the bsd gene present in the drug selection cassette on the plasmid. Integration into the hrp2 loci was detected in the pfk2 transfection culture only (panel C, lane h).