Generation of Pb<i>^pfpkg</i> parasites.

<p>A) Schematic diagram of the endogenous <i>pbpkg</i> locus, the targeting construct and the transgenic <i>pb^pfpkg</i> locus. Areas of 5′UTR and 3′UTR cloned into the targeting vector are indicated, S = spacer. Arrows 1–6 indicate binding sites for primers used in diagnostic PCR. Primers 1 and 2 were used to detect 5′ integration. Primers 3 and 4 were used to determine 3′ integration. Primers 5 and 6 bind specifically to the endogenous <i>pbpkg</i> and are used to confirm absence of the endogenous gene in the transgenic line. The area homologous to the probe used in Southern blotting and <i>Bcl</i>I restriction sites used for diagnostic digest are indicated. B) Diagnostic PCR used to determine integration of the targeting construct into the Pb<i>^pfpkg</i> transgenic line. C) Southern blot following <i>Bcl</i>I digest shows integration of the targeting construct as a specific 3.9 kb band and absence of the endogenous 5.1 kb band in the transgenic line (Pb<i>^pfpkg</i>) in comparison to wild type (WT-GFP). D) PFGE of wild type (WT-GFP) and mutant parasite (Pb<i>^pfpkg</i>) confirms integration into the correct chromosome. E) Western blot of asexual blood and ookinete stages confirm expression of PfPKG in the transgenic line (the transgenic PfPKG bands are 25.8% and 21.8% of the PbPKG band in the WT-GFP line in asexual blood and ookinete stages respectively).</p

Parasite development in the mosquito.

<p>A) Exflagellation in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. The mean number of exflagellation centres was 7.0 for wild type and 6.8 for Pb<i>^pfpkg</i> parasites. Bar, mean ± SEM (Mann-Whitney U test: ns, not significant, p>0.05 compared to WT-GFP). B) Ookinete conversion in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. Wild type conversion was 63% and Pb<i>^pfpkg</i> conversion was 10%. Data shown as mean ± SEM (Mann-Whitney U test: ***, p<0.001 compared to WT-GFP). C) Gut oocyst numbers in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. Wild type infected guts contained 100 oocysts and Pb<i>^pfpkg</i> infected guts with less than 10 oocysts. Bar, mean ± SEM (Mann-Whitney U test: ***, p<0.001 compared to WT-GFP).</p