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. 2015 Mar 23;290(19):12147–12164. doi: 10.1074/jbc.M115.637694

FIGURE 1.

FIGURE 1.

Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR (arrows 1 and 2) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP (primers 3+5) and wild type (primers 3+4). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 (c0) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR (arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP (primers 17+5) and wild type (primers 17+18). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei (blue) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).