Targeted replacement of the <i>L. major</i> and <i>L. infantum LRP</i> gene.

<p>Schematic drawing of the <i>LRP</i> locus with <i>Sal</i>I sites of <i>L. major</i> and <i>L. infantum</i> (A, C). (B) Southern blot analysis of <i>L. major</i> Friedlin genomic DNA digested with <i>Sal</i>I and hybridized with a 3′ UTR probe of <i>LRP</i> gene (a ∼500 bp fragment just downstream the stop codon of <i>LRP</i> gene) (upper panel) and then with a LRP PCR amplified gene fragment of ∼500 bp (bottom panel). (D) Southern blot analysis of <i>L. infantum</i> 263 genomic DNA digested with <i>Sal</i>I and hybridized with a 3′ UTR probe of <i>LRP</i> gene (a ∼500 bp fragment just downstream the stop codon of <i>LRP</i> gene) (upper panel) and then with the LRP PCR specific fragment (bottom panel). Molecular weight is indicated on the left. Lane 1, <i>Leishmania</i> WT cells; Lane 2, <i>Leishmania</i> SKO cells (LRP/NEO); 3, <i>Leishmania</i> DKO cells (NEO/NEO) at the <i>LRP</i> locus obtained by loss of heterozygosity. (E, F) SbIII susceptibilities of <i>L. major</i> and <i>L. infantum LRP</i> mutants and hybrid parasites. The EC₅₀ values were determined by culturing promastigotes parasites in the presence of increasing concentrations of SbIII. The average of three independent experiments is shown with a statistical significance observed by Student's t-test (p<0.05) (*).