Plasmid Integration for PfPrp2 Gene

To create the plasmid used for the integration of the RFA at the 3'end of the PfPrp2 gene by single crossover, a PCR fragment containing nucleotides 2819 to 3774 of PfPrp2 without a stop codon was cloned into the Xho1-AvrII sites of the pGDB vector [17 (link)]. Because wild-type P. falciparum is sensitive to trimethoprim, the DHFRdd system requires the use of parasite strains resistant to this antifolate. The system was developed with a PfPlasmepsin 1 knock out strain (ΔPM1) (PfPM1 is a non essential gene) expressing hDHFR, rendering the parasites resistant to TMP[17 (link)] so we decided to use this same strain for our studies. Parasites were transfected, and integrants were selected as described previously[48 (link)]. Briefly, P. falciparum 3D7 ΔPM1 parasites [49 (link)] were transfected with 100 ug of purified plasmid DNA (Promega). Positive selection for transfectants was achieved using 2.5 mg/ml BSD (Sigma-Aldrich) and 5 uM TMP (Sigma-Aldrich). Integration was monitored by Southern blots according to standard procedures.