Upper panel: IFA showing the subcellular localization of GAP45-HA3 to the periphery of intracellular merozoites in mock-treated GAP45:loxP parasites and the loss of GAP45-HA3 upon RAP treatment. Over 99% of schizonts examined by IFA were GAP45 and HA negative following RAP treatment. Bar, 5 mm. Rapamycin (RAP) treatment of synchronized GAP45:loxP parasites to induce DiCre activity resulted in the anticipated excision of the GAP45-HA3 gene completely abolished the IFA signals by the end of the erythrocytic cycle in which the parasites were RAP treated (cycle 0) This indicated rapid and efficient silencing of GAP45 expression within the course of a single erythrocytic cycle (~48 h), Lower panel: IFA showing continued expression of GAP45 from the modified Pfs47 locus following RAP-mediated silencing of GAP45-HA3 expression in GAP45-HA3:loxPcomp parasites. Bar, 5 mm. Treatment with RAP of the resulting parasite clone (called GAP45:loxPcomp) ablated expression of the HA-tagged floxed GAP45 gene as expected but did not ablate expression of GAP45 from the second gene copy. Collectively, these results convincingly demonstrated that GAP45 is indispensable for long-term survival of asexual blood-stage.

Perrin AJ, Collins CR, Russell MRG, Collinson LM, Baker DA, Blackman MJ. The Actinomyosin Motor Drives Malaria Parasite Red Blood Cell Invasion but Not Egress. MBio. 2018 Jul 3;9(4). pii: e00905-18.