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. 2011 Oct;157(Pt 10):2785–2794. doi: 10.1099/mic.0.049023-0

Fig. 2.

Fig. 2.

Attempt to disrupt the pfnek-1 gene. (a) Strategy for pfnek-1 gene disruption. The knockout vector contains a PCR fragment spanning positions 73–630 of Pfnek-1 coding sequence, excluding two kinase subdomains essential for activity. Single crossover homologous recombination results in a pseudo-diploid configuration with two truncated copies lacking essential catalytic residues. The location of PCR primers (numbered arrows) and the restriction sites used for Southern blot analysis are indicated. (b) PCR genotyping. Genomic DNA was isolated from transfected and parental 3D7 parasites, and subjected to PCR using the indicated primers (see Fig. 2a). Lanes: 1, primers 1+3 (diagnostic for 5′ integration event, 0.6 kb); 2, primers 2+4 (diagnostic for 3′ integration event, 1.5 kb); 3, primers 2+ 3 (diagnostic for the episome, 0.7 kb); 4, primers 1+ 4 (diagnostic for wild-type locus, 2.1 kb). Sizes of co-migrating markers are indicated in kb. (c) Southern blot analysis of transfected parasites. Genomic DNA digested with SpeI and NcoI was probed with the Pfnek-1 fragment used in the pCAM-BSD-Nek-1 vector (positions 73 to 630 of the coding sequence). Sizes of co-migrating markers are indicated in kb.