Generation of <i>P. falciparum</i> parasites lacking expression of P52.

<p>(A) Illustration of the DNA construct (m144) used for the targeted gene disruption of <i>p52</i> and the <i>p52</i>-genomic locus before and after integration. Shown are the p52 gene and target sequence (amplified using 1624 & 1625), the paralog of p52, p36, and the <i>T. gondii dhfr/ts</i> selection cassette. In addition, primer pairs and restriction sites for diagnostic PCR and Southern analysis are shown (see B and C). hrp – histidine rich protein. (B) Southern analysis of <i>BstN</i>I/<i>SnaB</i>I digested genomic DNA of Wt and <i>Δp52</i> demonstrates correct disruption of <i>p52</i>. DNA was hybridized with a <i>p52</i> specific probe detecting a 3.3 kb fragment in Wt, a 2.2 kb fragment for intact plasmid and the expected fragments of 1.3 kb and a 4.2 kb band (see A) in the two <i>Δp52</i> clones (<i>Δp52-1 and Δp52 -2</i>). (C) PCR analysis of genomic DNA of Wt and <i>Δp52</i> clones and the plasmid DNA (construct) demonstrates correct disruption of <i>p52</i>. Genomic DNA from Wt and <i>Δp52</i> asexual parasites and sporozoites was used as template for the PCR reactions. The Wt specific PCR was performed using primers 1638 and 1676 amplifying a 2.1 kb fragment. PCR primer pairs 1638 and L430, specific for integration of the DNA construct (see A) amplify a 2.0 kb fragment. Primer pairs 190 and 191 amplifying a 1.8 kb fragment from <i>T. gondii dhfr/ts</i> were used as a control.</p

Invasion capacity of Wt and Δ<i>p52</i> sporozoites in primary human hepatocytes <i>in vitro</i>.

<p>(A) Intra (In) and extracellular (Ex) sporozoites 3 hrs after incubation of sporozoites with primary human hepatocytes in culture. Sporozoites were first stained with anti-PfCSP antibodies (red). Then cells were permeabilised and sporozoites were stained with anti-PfCSP antibodies (green). Consequently, extracellular sporozoites will stain red AND green and intracellular sporozoites will stain only green. Nuclei of the hepatocytes (white arrow heads) were stained with DAPI (B) The percentage of intracellular/invaded sporozoites (Wt and Δ<i>p52</i> mutant lines) in primary human hepatocyte 3 hours after sporozoite incubation, as determined in the double anti-CSP staining immuno-fluorescence assay (see A). (C) The number of schizonts detected by IFA using anti-HSP70 antibodies and the nuclear dye DAPI formed 3 days after incubation with either Wt or Δ<i>p52</i> mutant sporozoites. (D) The number of schizonts detected by IFA using anti-HSP70 antibodies and the nuclear dye DAPI formed 5 days after incubation with either Wt or Δ<i>p52</i> mutant sporozoites.</p