Construction and analysis of fluorescent <i>P. cynomolgi</i> using a novel centromere construct.

<p>(A) Dot matrix analysis of a <i>P. cynomolgi</i> and <i>P. vivax</i> putative centromere (PCEN). Graphical representation of a matrix analysis of a <i>P. cynomolgi</i> PCEN aligned against itself (<i>left</i>), <i>P. cynomolgi</i> PCEN against the <i>P. vivax</i> PCEN (<i>middle</i>) and <i>P. vivax</i> PCEN aligned against itself (<i>right</i>). The analysis was performed using Dotlet <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888-Junier1" target="_blank">[46]</a> as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888-Iwanaga1" target="_blank">[22]</a>. The diagonal line within each analysis represents sequence identity, and the diagonal line indicates repetitive regions within each PCEN. Note the absence of the diagonal in the repetitive regions of the <i>P. cynomolgi</i> and <i>P. vivax</i> alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone-0054888-g001" target="_blank">Figure 1</a>, <i>middle</i> panel). (B) Schematic representation of the pPcyC-PAC-GFP_hsp70-mCherry_ef1α plasmid. The plasmid contains the <i>Tgdhfr-ts</i> selectable marker that confers resistance against pyrimethamine and two expression cassettes for constitutive expression of GFP and mCherry. Additionally, to maintain the plasmid throughout the life cycle, a putative <i>P. cynomolgi</i> centromere (PcyCEN) is included. (C) Schematic representation of the procedure used for transfection and analysis of <i>P. cynomolgi</i>. (D) PCR amplification of <i>gfp</i> and <i>mCherry</i> in PcyC-PAC-GFP_hsp70-mCherry_ef1α (PcyC-PAC) blood stage parasites. Wild type gDNA of <i>P. cynomolgi</i> M served as negative control. For a control PCR primers for the <i>circumsporozoite protein (csp)</i> were used. For primers used, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888.s001" target="_blank">Table S1</a>. (E) GFP and mCherry expression throughout the life cycle of <i>P. cynomolgi.</i> GFP and mCherry expression in pPcyC-PAC-GFP_hsp70-mCherry_ef1α transfected <i>P. cynomolgi</i> blood stage parasites (a ring and a trophozoite or gametocyte), in oocysts 5 days post mosquito feeding and in salivary gland sporozoites 12 days post feeding. In the Brightfield panel two salivary gland lobes can be distinguished; only one lobe contains sporozoites. In the panel on the right GFP and mCherry expression is shown in Hoechst 33342 stained day 6 liver stages. Note the autofluorescence of hepatocytes in the GFP channel in contrast to the mCherry channel. A small uninucleate (arrow) and a large multinucleate liver stage are visible, confirmed by staining of fixed parasites with anti-HSP70 antibodies (<i>lower right panel</i>). White bars correspond to 10 µm (blood and mosquito stages) and 50 µm (liver stages).</p

Flow cytometry and cell sorting of <i>P. cynomolgi</i> liver stage parasites, including hypnozoite-forms.

<p>(A) Liver stage parasites used for flowcytometry as detected by anti-HSP70 antibodies 3 days and (B) 6 days post hepatocyte infection. White bars correspond to 50 µm. Note that day 3 cultures contain uniform small parasites while day 6 cultures contain both small and large liver stages (arrows). Flow cytometric plots of PcyC-PAC-GFP_hsp70-mCherry_ef1α (PcyC-PAC) <i>P. cynomolgi</i> liver stage parasites show a single GFP positive population compared to wild type parasites 3 days post hepatocyte infection (A, Gate 1) and two GFP positive populations 6 days post hepatocyte infection (B, Gates 2 and 3). The y-axis represents the PE-Texas Red Channel (for detection of autofluorescence), while the x-axis represents the GFP signal. (C) Post-sorting images of PcyC-PAC-GFP_hsp70-mCherry_ef1α<i>P. cynomolgi</i> liver stage parasites ‘GFPlow’ (Gate 2) and ‘GFPhigh’ (Gate 3) parasites sorted at day 6 post hepatocyte infection. The upper panel shows a GFP/Brightfield overlay while the lower panel shows mCherry/Brightfield overlay. The panels below show close-ups of the sorted parasites revealing the size differences between the ‘GFPlow’ and ‘GFPhigh’ populations. White bars correspond to 50 µm.</p