Mutant/mutation
The mutant expresses nanoluciferase under control of the (ookinete specific) ctrp promoter. The nanoluciferase expression cassette has been integrated into the neutral 230p locus.

The mutant has been generated and selected using the GIMO transfection method ('gene insertion/marker out') of transfection of a GIMO mother line that contains the hdhfr::yfcu selectable marker into the silent 230p locus.

The mutant has been generated in the reference line GIMO_PbANKA (RMgm-687). This GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice

Protein (function)

Phenotype
Ookluc had negligible nLuc activity in circulating blood stages (time 0, no incubation), and in in vitro fertilization assays (conversion) the signal has a relative increase of ~40-fold  after 6 hr and ~2,000-fold after 24 hr, demonstrating that the ctrp promoter is sufficiently silenced to prevent detectable nLuc expression in blood stages and is activated only upon conversion into mosquito sexual stages.

To verify if the increase in nLuc activity in (ookinete) conversion assays with Ookluc was  dependent on fertilization, we deleted the mtrap gene from the Ookluc genome. MTRAP-knockout (MTRAP-KO) gametocytes can activate, but the gametes remain trapped inside the host cell. There was no increase of nLuc activity in Ookluc-MTRAP-KO parasites after 24 hr of conversion assay – the absence of ookinetes was confirmed by blood smears – showing that gametocyte activation per se is not sufficient for activation of the ctrp promoter, hence nLuc expression and activity in Ookluc parasites depend on gamete egress and fertilization.

The increase in nLuc activity after 6h of conversion assay with Ookluc showed that the ctrp promoter likely becomes active soon after zygote formation. Inhibition of palmitoylation with 2-Bromopalmitic acid (2-BP) 1 hour post Ookluc activation, blocking zygote to ookinete morphogenesis, did not reduce the nLuc activity after 6h or 24h. This indicates that once the ctrp promoter is activated in zygotes it accumulates nLuc regardless of ookinete  formation, and precludes Ookluc as a tool for searching compounds with TB activity through inhibition of ookinete morphogenesis.

Additional information
From the Abstract:
'Here we demonstrate the generation of a Plasmodium model for drug screens against  gametes and fertilization. The new P. berghei line, named Ookluc, was genetically and pharmacologically validated and scalable for HTS. Screening the Pathogen Box from the Medicines for Malaria Venture (MMV) using the new model identified promising TB compounds. The use of Ookluc in different libraries of compounds may aid in the identification of transmission-blocking drugs not assessed in screens against asexual stages or gametocytes.'

Other mutants