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. 2011 Sep 1;7(9):e1002197. doi: 10.1371/journal.ppat.1002197

Figure 3. Generation of pyrom1(-) parasites by allelic exchange.

Figure 3

(A) Schematic of targeting strategy using the targeting vector pR1KO which has the pbDHFR/TS-GFP selection cassette flanked by a 5′homologous arm (600 bp) and a 3′ homologous arm (600 bp) corresponding to the 5′ and 3′ regions of the pyrom1 gene, respectively. After double cross over homologous recombination the endogenous pyrom1 gene is replaced by the selection cassette resulting in parasites deficient for pyrom1 (R1KO). (B) Proper gene replacement was verified by Southern Blot analysis in two knockout clones (KO1, KO2). Strategy for enzyme restriction digestion with either ScaI (S) or a combination of ScaI and MscI (M) using a probe (dumbbell bar) specific for the 3′ homologous arm is depicted in (A). A ScaI fragment that corresponds to the wildtype (Wt) genomic locus migrates at ∼7000 bp in both blots. Digestion of the recombinant locus (R1KO) with either ScaI/MscI or ScaI reveals expected bands migrating at the expected sizes of ∼4000 bp and ∼12,000 bp, respectively. (C) Lack of pyrom1 gene expression in R1KO clones from mixed blood stages was verified by RT-PCR. Analysis of pyADA expression, an unrelated gene, was used as a positive control. Expression of pyrom1 was analyzed using primers, R1-F and R1-R, which amplify the Open Reading Frame (ORF) of pyrom1. The expected pyrom1 ORF band (expected size ∼800 bp) is readily amplified from wildtype (WT) cDNA, but is not amplified from cDNA of the two pyrom1(-) clones (KO-1 and KO-2).