Figure 2. TgPI3-Kinase conditional knock-in by promoter exchange strategy.

(A) Schematic representation of the strategy used to replace the endogenous promoter of TgPI3K with the tetracycline-inducible promoter. The 5′TgPI3KUTR-DHFR-tetO7-SAG4-HA(2)NtTgPI3K plasmid contains the TgPI3K 5′ UTR (in black), the dihydrofolate reductase (DHFR) cassette (in blue) and the N-terminal genomic coding sequence of TgPI3K gene (grey) fused to two HA tags (red) under the control of the inducible tetO7SAG4 promoter (orange arrow). Black arrows represent the primers used for PCR analysis and the length of the PCR fragments is indicated. (B) PCR analysis performed on pi3ki confirming double homologous recombination. Genomic DNA from TATi1-ku80ko parasites was used as negative control. (C) Schematic representation of the crossover event that will result in the replacement of TgPI3K tetracycline inducible locus by the PI3-Kinase gene present in the recombineering modified ToxP331 cosmid in fusion with a 3 HA epitope at the 3′ end of the TgPI3K gene. CAT: chloramphenicol-acetyl-transferase. (D) Semi-quantitative RT-PCR analysis of TgPI3K expression in the wild-type, mutant and complemented parasites, preceded or not by three days of induction by ATc to regulate expression. Primers specific of the gene coding for FYVE1 were used as a loading control. (E) DD-FYVE(2)-GFP expressing pi3ki parasites were processed for IFA ± ATc and in the presence of Shield-1. The DD-FYVE(2)-GFP protein shows a punctuate staining in pi3ki parasites non-treated with ATc. A cytosolic labelling was observed when the pi3ki parasites were cultured in the presence of ATc. (F) Plaque assay of HFF monolayer infected with TATi1-ku80ko or pi3ki or pi3kiC parasites pre-treated first during 48 h with ATc. After 7 days ± ATc, the HFF were stained with Giemsa.