Figure 2: Experimental genetics approaches employed to study 35 MTP.

(a) Schematic representation of the generation of mtp⁻ and mtp::tag parasites. The 5′ and 3′ flanking regions of the target genes were cloned adjacent to the selection cassette resulting in the gene deletion transfection vector (pMTP-KO). For endogenous tagging, the carboxy terminus was cloned in frame with an mCherry-3xMyc tag and the 3′FR was cloned distal of the selection cassette, resulting in the endogenous tagging vector (pMTP-tag). By double crossover homologous recombination the targeted MTP was predicted to be either replaced or endogenously tagged with the fluorescent tag, respectively. (b) Schematic representation of the P. berghei transfection protocol adapted from Matz and Kooij³⁷. Cultured and synchronized schizonts are transfected and successfully modified parasites are selected in vivo using pyrimethamine. When the parasitaemia is 0.1–1.0%, 50 isogenic mutant parasites are isolated by flow cytometry. (c) Schematic overview of the standardized phenotypic profiling protocol of four life cycle checkpoints looking at (1) blood-stage growth in the mouse, (2) exflagellation rates as a measure of mouse-to-mosquito transmission, (3) salivary gland sporozoite numbers, and (4) prepatency following infections by natural bites to follow mosquito-to-mouse transition and ability to complete the full life cycle.