Schematic images of the centromere region of <i>P</i>. <i>vivax</i> and <i>P</i>. <i>yoelii</i> used in this study and the design of the plasmids to evaluate centromere function.

(A) The P. vivax centromere sequence was selected from chromosome 11 (CM000452.1). DNA fragments of approximately 1.9 and 1.5 kb size were used for plasmid construction and named PvCEN11S2 and PvCEN11S3, respectively. The P. yoelii centromere sequence was selected from chromosome 5 (DQ054838.1) and approximately 1.8 kb DNA fragment was used for the plasmid, named PyCEN5. These sequences include repeat sequence motifs (white arrowhead). (B) Centromere plasmids evaluated in this study (PvCEN11S2-H86HD-TG, PvCEN11S3-H86HD-TG, and PyCEN5-H86HD-TG) and a control plasmid without a centromere region (pNoCEN). CEN, centromere region; 5' PfHSP86, the 5' untranslated region (UTR) of P. falciparum heat shock protein 86; hDHFR, human dihydrofolate reductase open reading frame; 3TyGFP, triple Ty1 tag and green fluorescent protein open reading frame; 3' PbDT, 3' UTR of P. berghei dihydrofolate reductase-thymidine kinase; and 5' PvHSP86, 5' UTR of P. vivax heat shock protein 86.</p

Segregation efficiency at the end of multiplication periods.

Segregation efficiency at the end of multiplication periods.</p

Growth curve and the percentages of GFP-positive parasites.

(A) Infected red blood cells (1 x 106) of transgenic parasites (PvCEN11S2, PvCEN11S2-H86HD-TG; PvCEN11S3, PvCEN11S3-H86HD-TG; PyCEN5, PyCEN5-H86HD-TG; and pNoCEN) or the parental parasite (17XL) were inoculated into each mouse and maintained with pyrimethamine except 17XL. Parasitemias were determined daily until day 7. (B) The percentage of GFP-positive parasites without pyrimethamine was examined at 7 time points until day 28. Mean and standard deviation from 5 mice were plotted for each group.</p

<i>P</i>. <i>vivax</i> promoter activities in <i>P</i>. <i>yoelii</i>.

Firefly luciferase was expressed under a panel of P. vivax promoters. P. falciparum heat shock protein 86 promoter was used as a positive control, because this promoter has been shown to be active in P. yoelii. A plasmid without a promoter sequence upstream of the luciferase open reading frame was used as a negative control. Luciferase activities from purified schizont stage parasites were normalized by the plasmid copy number of each parasite quantitated by qPCR. Results from two independent experiments are shown. Mean and standard deviation from triplicate wells are plotted.</p

Change in plasmid copy numbers 28 days after removal of pyrimethamine.

Plasmid copy numbers were quantitated before (day 0) and after (day 28) removal of pyrimethamine drug pressure in transgenic parasites with PvCEN11S2-H86HD-TG (PvCEN11S2), PvCEN11S3-H86HD-TG (PvCEN11S3), PyCEN5-H86HD-TG (PyCEN5), or pNoCEN. Three or five mice were used for each group on days 0 and 28, respectively. After the one-way ANOVA test indicated significant difference, Tukey's post-hoc multiple comparisons test was performed. Significant difference was indicated with p values. n.s. indicates not significant (p ≥ 0.05).</p

NanoLuc luciferase activities from various stages of transfected parasites with pigHD_vHSP70-1k-NLuc-mCherry.

Clones 5 and 15 are cloned transfectants with pigHD_vHSP70-1k-NLuc-mCherry. P. yoelii 17XL was used as a negative control. Relative luciferase activities (relative light units, RLU) from 105 blood-stage parasites before and after mosquito and liver stage development (A), 102 mosquito midgut oocysts (B), or 103 mosquito salivary gland sporozoites (C) are shown. Mean and standard deviations from triplicate wells are plotted.</p

Percentage of GFP-positive oocysts and blood-stage parasites before and after mosquito and liver stage development.

Oocyst numbers (A) and the percentage of GFP-positive oocysts (B) were examined for 5 mosquitoes on day 11 after feeding of mice infected with transgenic parasites with PvCEN11S2-H86HD-TG (PvCEN11S2), PvCEN11S3-H86HD-TG (PvCEN11S3), PyCEN5-H86HD-TG (PyCEN5), pNoCEN, and the parental parasite 17XL. Two independent experiments were performed for PvCEN11S2 and PvCEN11S3 transgenic parasites. (A) Black circles and bars indicate the median and distribution of oocyst numbers in each mosquito. (C) Percentage of GFP-positive blood-stage parasites were examined on the feeding day (day 3 after pyrimethamine removal) and after mosquito and liver stage development (day 30 after pyrimethamine removal).</p

mCherry signals from blood-stage parasites, mosquito midgut oocysts, salivary gland sporozoites, and blood stages after liver stage development of transfected parasites with pigHD_vHSP70-1k-NLuc-mCherry.

(A) Schematic image of pigHD_vHSP70-1k-NLuc-mCherry, a piggyBac-based plasmid expressing NanoLuc® Luciferase-mCherry fusion protein under the pvhsp70 promoter (~1 kb). 5' PvHSP70, the 5' untranslated region (UTR) of P. vivax heat shock protein 70; 3' PbDT, 3' UTR of P. berghei dihydrofolate reductase-thymidine kinase; 5' PfCAM, 5' UTR of P. falciparum calmodulin; hDHFR, human dihydrofolate reductase open reading frame; 3' PfHRP2, 3' UTR of P. falciparum histidine-rich protein 2; ITR, inverted terminal repeat sequences for the piggyBac transposon system. (B) Mice were infected with transgenic parasites with pigHD_vHSP70-1k-NLuc-mCherry or the parental parasite 17XL by intravenous injection of 106 infected red blood cells and maintained without pyrimethamine. Three days later thin blood smears were made (day 3) and mosquitoes were fed. Mosquitoes were dissected on day 11 after feeding (day 14 after pyrimethamine removal) to count the number of the midgut oocysts and to observe mCherry signals. Whole salivary glands were examined to detect mCherry signals on day 18 (day 21 after pyrimethamine removal). The remaining mosquitoes were dissected to collect salivary gland sporozoites on day 24, which were then injected intravenously into mice. Parasites appeared in the blood on day 6 after inoculation (day 30 after pyrimethamine removal) and were observed for mCherry signals. Parasite nuclei were stained with Hoechst (Hx). DIC, differential interference contrast image. Bar indicates 10 μm.</p

GFP signals from mosquito midgut oocysts, salivary gland sporozoites, and blood stages after liver stage development of transfected parasites.

Forty mosquitoes were fed on day 3 after intravenous injection of 106 infected red blood cells into mice for each group (transgenic parasites with PvCEN11S2-H86HD-TG (PvCEN11S2), PvCEN11S3-H86HD-TG (PvCEN11S3), PyCEN5-H86HD-TG (PyCEN5), or pNoCEN and the parental parasite 17XL) and were dissected to count the numbers of midgut oocysts and to observe GFP signals on day 11 after feeding (day 14 after pyrimethamine removal). Whole salivary glands were examined to detect GFP signals on day 18 (day 21 after pyrimethamine removal). Ten to fifteen remaining mosquitoes were dissected to collect salivary gland sporozoites on day 21 after feeding (day 24 after pyrimethamine removal). Sporozoites were counted and observed for GFP signals. All possible collected sporozoites were injected intravenously into mice and parasites that appeared in the blood on day 6 after inoculation (day 30 after pyrimethamine removal) were observed for a GFP signal. Parasite nuclei were stained with Hoechst (Hx). DIC, differential interference contrast image. Bar indicates 10 μm.</p