Figure 1. Replacement of pfpkg intron 3 with 2loxPint allows normal PKG expression and parasite replication.

(A) Shown is the integrated 2loxPint sequence derived by nucleotide sequencing of genomic DNA from the P. falciparum pfpkg_2lox line, aligned with the expected sequence (both in red). The loxN and lox2272 sites are in grey boxes, with the unique internal AccI site underlined. Boundaries of the 3′ end of exon 3 (AAAG) and the 5′ end of exon 4 (GGT) are shown in black. (B) Schematic representation of the strategy used to replace intron 3 of the pfpkg gene with 2loxPint in the DiCre-expressing B11 P. falciparum line. Positions of oligonucleotides used for diagnostic PCR are indicated (red and blue arrows). (C) (Left) Diagnostic PCR results showing absence of the endogenous intron 3 in the pfpkg_2lox line (2lox) relative to the parental B11 line (wt). (Right) Diagnostic PCR results and restriction digest of the PCR amplicon with AccI, showing the expected digestion only of the amplicon from the pfpkg_2lox line (2lox). (D) Western blot analysis of extracts of wt and pfpkg_2lox schizonts, showing similar expression levels of PKG (∼98 kD, indicated). Antibodies to the cytoplasmic parasite protein HSP70 were used as a loading control. (E) Growth curves showing replication of DMSO-treated (control) or RAP-treated pfpkg_2lox parasites relative to the parental line B11. Mean values are shown from triplicate experiments. Error bars ± SD (n = 3).