Pbtert deletion and selection of tert- mutants.

<p>(A) Schematic representation of the construct used to delete the <i>tert</i> gene. The construct, containing the <i>Tgdhfr-ts</i> selectable marker (SM) cassette, targets the <i>tert</i> gene at the flanking regions (red) by double cross-over integration. The red arrows indicate primers used for diagnostic PCR to confirm correct disruption of <i>tert</i>. Boxes correspond to lanes on the PCR gels in (B), (D) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108930#pone.0108930.s001" target="_blank">Fig. S1A</a>. (B) Diagnostic PCR of uncloned parasites transfected with a DNA construct to delete the <i>tert</i> gene. Parasites were collected and analysed directly after transfection and selection with pyrimethamine (parent populations). Diagnostic PCR shows the presence of parasites with correct disruption of the <i>tert</i> gene. In all experiments (1065, 1078, 1138, 1207, 1217) the 5′ and 3′ integration fragments (lanes 5′, 3′), as well as the <i>Tgdhfr-ts</i> fragment (lane SM) were amplified. However, all populations contained parasites with a wild type <i>tert</i> gene as shown by amplification of the wild type <i>tert</i> fragment (lane wt). The primer pairs used are shown in (A) and expected fragment sizes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108930#pone.0108930.s004" target="_blank">Table S2</a>. <i>pbs21</i>-specific primers were used as a positive control for all the PCR reactions (“+”). The water control is marked as “-“. (C) Southern analysis of separated chromosomes using the 3′UTR <i>Pbdhfr-ts</i> probe shows only in experiment 1065 and 1217 hybridisation with chromosome 14 on which the <i>tert</i> gene is located. This probe recognizes the endogenous <i>Pbdhfr-ts</i> gene on chromosome 7 in all populations and additional chromosomes in experiments 1078, 1138, 1207 (possible episomal construct signal). (D) Diagnostic PCR of uncloned and propagated parasites transfected with a DNA construct to delete the <i>tert</i> gene. The parent parasite populations of experiment 1065, 1207 and 1217 [see (B)] were propagated in mice (m0 = mouse 0, m1 = mouse 1) for another 1–2 weeks. Parasite populations collected were analysed by diagnostic PCR for the presence of parasites with correct disruption of the <i>tert</i> gene [primers same as in (B)]. In all populations no parasites with a disrupted <i>tert</i> gene could be detected by diagnostic PCR after 1 week (1207 all populations) or after two weeks of propagation (1065 uncl.2, 1217 uncl.2 m0 and 1217 uncl.2 m1).