Fig 1.

(A) Cartoon of the predicted P. falciparum PP7 protein showing the positions of the individual domains. Amino acid numbers are shown in black. The arrow indicates the point at which the protein product is truncated when the modified locus is excised in the transgenic parasites. (B) Schematic representation of the SLI strategy (23) used to produce the PP7-HA:loxP DiCre line and resultant RAP-induced disruption of the modified gene. Double-headed arrows represent the regions amplified by PCR in (C). Red arrowheads, loxP sites, yellow lollipops, translational stop codons, white box, phosphatase domain, light blue box, regions of re-codonized sequence (R.R.). (C) Diagnostic PCR analysis of genomic DNA (gDNA) from a transgenic PP7 parasite line verifying successful modification of target loci by SLI to produce PP7-HA:loxP. Efficient excision of “floxed” sequences is observed upon treatment with RAP. Track C represents the amplification of a control locus (PKAr) to check gDNA integrity. PCRs 1–4 are represented in the schematic locus in panel 1B. PCR 1 screens for the WT locus, PCR 2 for 5′ integration, PCR 3 for the excision of the “floxed” sequence, and PCR 4 for 3′ integration. See Table 1 for sequences of all primers used for PCR. Sizes for expected amplification products are as follows: C, control locus (primers 5 and 6) 836 b.p. PCR 1 (primers 7 and 8) 1,296 b.p, PCR2 (primers 7 and 9) 3,559 b.p., PCR3 (primers 7 and 10) 1,362 b.p. (RAP) and PCR 4 (primers 11 and 8) 1,013 b.p. (D) Western blot analysis of expression [dimethyl sulfoxide (DMSO), control] and ablation (RAP) of PP7-HA from highly synchronous mature schizonts. The expression of GAPDH (PF3D7_1462800) is shown as a loading control. (E) Immunofluorescence analysis (IFA) analysis showing the diffuse peripheral localization of PP7-HA and the loss of expression upon RAP treatment (16 h post-invasion). Over 99% of all RAP-treated PP7-HA:loxP schizonts examined by IFA showed diminished HA expression in three independent experiments. Scale bar, 2 µm.