Fig. 1

Efforts for disruption of the Pfdut gene by single crossover recombination. a After a single crossover event of the plasmid with the dut locus, the KO construct would lead to the generation of two truncated inactive copies of the gene. b The KOkon plasmid should generate a functional copy of the dut gene and a non-functional pseudogene upon single crossover recombination. c Southern blot analysis of the pHH1-dutKO transfected line after drug cycles. Genomic DNA of the cell line transfected with pHH1-dutKOk after two (lane 1) and three (lane 2) cycles of drug pressure, genomic DNA of the 3D7 parental line (lane 3) and 5 ng of purified plasmid DNA (lane 4) were digested with NdeI. The 1.8 kb band corresponding to the endogenous locus is present in the three genomic DNAs, whereas the episome (5.4 kb) is present only in lanes 1 and 2. d Southern blot analysis of the pHH1-dutKOkon transfected line after drug cycles. Genomic DNA of the 3D7 parental line (lane 1), the cell line transfected with pHH1-dutKOkon after two (lane 2) and three (lane 3) cycles of drug pressure and 5 ng of purified plasmid DNA (lane 4) were digested with NdeI. The 1.8 kb band corresponding to the endogenous locus is present in the three genomic DNAs, whereas the episome (5.4 kb) is present only in lanes 2 and 3. In lanes 2 and 3, two new extra bands of 2.2 kb and 5 kb were detected; the intensity of the bands increased from cycle 2 to cycle 3, corresponding to an enrichment of the culture in parasites with the plasmid integrated in the dut locus