<i>PbLplA1</i> is essential for propagation of asexual stages.

<p><i>A.</i> Insertion strategy to generate <i>LplA1</i> null mutants. The <i>PbLplA1</i> genomic locus (I.) is targeted with a <i>Hpa</i>I-linearized targeting vector (b3D-LplA1-int1, II.) containing 5′ and 3′ truncations of the <i>LplA1</i> open reading frame and the <i>T. gondii DHFR/TS</i> positive selectable marker. Upon a single cross-over event, the region of homology is duplicated, resulting in two truncated, non-expressed <i>LplA1</i> copies in the recombinant locus (III.). Wild-type, plasmid, and integration-specific primer combinations are indicated by arrows and sizes of expected fragments are shown. <i>B.</i> Genotyping indicates absence of successful integration. While episomal plasmid (primer pair b3D-1 and b3D-2 amplifying a 1.63 kb pair; lane 3) and the endogenous <i>LplA1</i> gene (primer pair Pb-10 and Pb-11 amplifying a 2.48 kb fragment; lane 4) are amplified, no integration-specific bands (lane 1 and 2: 3′- and 5′-specific integrations, respectively) using the primer pairs b3D-1 and Pb-11 to amplify a 2.38 kb 3′-fragment and Pb-10 and b3D-2 to amplify a 2.45 kb 5′-fragment were obtained from each of 3 transfected parasite populations.