Genotypic analysis of P. falciparum transfected with the complementation construct pHH1-LplA1-KOkon.

<p><i>A</i>. Schematic representation of endogenous <i>PfLplA1</i> gene locus (I.), transfection plasmid pHH1-<i>LplA1</i>-KOkon (II.) and <i>PfLplA1</i> gene locus after single cross-over recombination (III.). The restriction enzyme used for diagnostic digest is shown (<i>Nde</i>I) and the expected sizes of diagnostic bands after hybridization with <i>PfLplA1</i> or <i>hDHFR</i> are indicated. <i>B</i>. Southern blot analyses of transfected parasite lines. Genomic DNA of wild-type and <i>LplA1</i>-KOkon parasites was digested with <i>Nde</i>I and probed with the <i>PfLplA1</i> ORF (left panel). In all parasite lines analysed, the endogenous gene is present (1.9 kb band) and in lanes 3 and 4 two additional DNA fragments are detected by the probe (in lane 2 less DNA was loaded on the gel so that the plasmid band is hardly visible). The faint 6 kb band is diagnostic for the presence of the transfection plasmid (see scheme), but the band of ∼9 kb (*) cannot be assigned to any specific integration event. Lane 1, wild-type D10; lane 2, <i>PfLplA</i>-KOkon, cycle 1; lane 3, <i>PfLplA</i>-KOkon, cycle 2; lane 4, <i>PfLplA</i>-KOkon, cycle 3. The same blot was stripped and re-probed with the probe specifically detecting the selectable marker <i>hDHFR</i> and apart from faint plasmid bands at 6 kb in lanes 2, 3 and 4 two additional bands of ∼9 kb (*) and 0.5 kb (*) are detected. Lane 1, wild-type D10; lane 2, <i>PfLplA</i>-KOkon, cycle 1; lane 3, <i>PfLplA</i>-KOkon, cycle 2; lane 4, <i>PfLplA</i>-KOkon, cycle 3. <i>C.</i> Genotyping by pulsed field gel electrophoreses. Chromosomes of wild-type <i>P. falciparum</i> D10 (lane 1) and parasites transfected with the pHH1-<i>LplA1</i>-KOkon construct in cycle 0 (lane 2) and cycle 3 (lane 3) were analysed by PFGE and the blots were probed with the <i>PfLplA1</i> open reading frame (left panel) or with the <i>hDHFR</i> open reading frame (right panel) present in the transfection plasmid. The <i>PfLplA1</i> probe detected the endogenous <i>PfLplA1</i> gene locus on chromosome 13 in wild-type and transfected parasites. However, the probe also detected a strong signal at the bottom of the blot where the non-separated chromosomes <10 are running. The <i>hDHFR</i> probe similarly generated signals on chromosome <10, but there is no sign of integration into the <i>LplA1</i> gene locus on chromosome 13.