Figure 6
Disruption of the Pfnek-4 gene. A, Strategy for gene disruption. The schematic representation shows the Pfnek-4 locus, the gene-targeting construct used for gene disruption by homologous recombination, and the pseudo-diploid locus resulting from integration of the KO construct. The primers used for the diagnostic PCRs are indicated. BSD, blasticidin-resistance cassette. B, PCR analysis indicating disruption of the Pfnek-4 gene in parasite clones cl3 and cl4 obtained from two independent transfections. Genomic DNA isolated from 3D7 wild-type parasite and Pfnek-4- clones cl3 and cl4, were subjected to PCR using the indicated primers OL-761 and OL-763 (diagnostic for the wild-type locus), OL-761 and OL-168 (diagnostic for 5’ integration), and OL-167 and OL-763 (diagnostic for 3’ integration). PCR products corresponding to expected sizes for wild-type (1288-bp), 5’ integration (1179-bp) and 3’ integration (1277-bp) are indicated on the right. C, Giemsa-staining of thin smears of pfnek-4- gametocytes at days 12–14 of in vitro tissue culture stimulating gametocyte production, showing the presence of stage IV (left panel) and stage V (right panel) gametocytes. Scale bars, 5.0 μm.