Pf332 transfection constructs and integration events. (A) Schematic representation of the pHTKΔ332 transfection plasmid and disruption of the Pf332 gene (3D7 Pf332) in P falciparum 3D7 parasites. The proposed model of the Pf332 gene locus disrupted by incorporation of the hdhfr resistance cassette in drug-resistant transgenic KO parasites is also shown (3D7ΔPf332). The position of relevant restriction enzyme sites for BamHI (B), the expected sizes of restriction fragments, and the position of the hdhfr probe (gray bar) are shown. (B) Schematic representation of the pHC1-332Δa and pHC1-332Δb transfection plasmids and the site of targeted disruption of the Pf332 gene (3D7 Pf332) in 3D7 parasites. The proposed models of integration of both plasmids into the Pf332 gene (3D7ΔPf332a/3D7ΔPf332b) are also shown. The position of relevant restriction enzyme sites for XbaI (X) and the expected sizes of restriction fragments are shown. (C) Southern blot analysis of BamHI-digested pHTKΔ332 and genomic DNA from 3D7 and the 4 3D7ΔPf332 KO clones: 11G7, 12F3, 13E2, and 13F7. Hybridization of the Pf332-F2 and hdhfr probes to digested DNA from all 3D7ΔPf332 parasite clones revealed restriction fragment sizes consistent with the disruption of Pf332 by double crossover homologous recombination and incorporation of the hdhfr drug resistance cassette. (D) Southern blot analysis of XbaI-digested pHC1-332Δa, pHC1-332Δb, and genomic DNA from 3D7 and the 3D7ΔPf332a (3F3 and 12H9) and 3D7ΔPf332b (5G1 and 13F5) clones. Hybridization of the Pf332a and Pf332b probes to digested DNA from the 3D7ΔPf332a and 3D7ΔPf332b clones, respectively, revealed restriction fragment sizes consistent with the disruption of Pf332 by single crossover homologous recombination and incorporation of the entire transfection plasmids.