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.

Abstract

(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