FIGURE 2:

Ablation of CpASP1 and conditional deletion of CpASP2 using rapamycin-inducible DiCre. (A) Fecal luminescence measurements from mice infected with parasites transfected with ∆CpASP1- and ∆CpASP2-targeting constructs. (B) Genetic strategy to replace a region of the CpASP2 gene that encodes the catalytic domain region with a recodonized sequence, including an HA epitope tag and flanking loxP sites (black triangles). The internal loxP site was placed within an intron. Nanoluciferase (Nluc) and neomycin resistance (Neo^R) markers were inserted downstream of the gene. The resulting CpASP2_loxP transgenic was crossed with a DiCre parasite (Shaw et al., 2024), and dual drug selection was used to isolate CpASP2_loxP DiCre line. (C) PCR on WT and CpASP2_loxP genomic DNA. Control (+) primers amplified the thymidine kinase gene, 5′ and 3′ integration regions indicated in B. (D) PCR on CpASP2_loxP parental and CpASP2_loxP DiCre lines using primers (from B) that span the floxed region. Addition of rapamycin leads to excision as indicated by the diagnostic 0.4 kbp band. (E) Representative micrograph of CpASP_loxP DiCre parasites treated with vehicle or rapamycin for 24 or 48 h. Loss of HA staining (green) is apparent at both timepoints. VVL in red, Hoechst in blue; scale bar, 5 µm. (F) The percentage of HA-positive parasites quantified from E. Each data point represents counts from one field of view. ****p<0.0001, one-way ANOVA. Data are shown as mean ± SD.