Figure 1

Pfmdr2 and Pfmdr5 deletion strategy and genotyping of the mutants. (A + B) Schematic diagrams showing the Pfmdr2 and Pfmdr5 gene deletion strategy. Following separate transfection and positive selection on WR99210, the pHHT-FRT-(GFP)-Pfmdr2 or pHHT-FRT-(GFP)-Pfmdr5 construct integrated into either the 5′ or 3′ target region (TR) in the NF54 wild type locus. Upon negative selection with 5-fluorocytosine, the mdr gene (black arrow) was replaced by the positive selection cassette harbouring the hdhfr::gfp fusion gene (green arrow) flanked by FRT sites (blue triangles) (PfΔmdr2gfp or PfΔmdr5gfp). This fusion gene was subsequently removed by FLPe-mediated excision upon pMV-FLPe transfection resulting in two mdr deletion lines, PfΔmdr2 and PfΔmdr5. cam: calmodulin; hdhfr::gfp: human dihydrofolate reductase fused to green fluorescent protein; hrp: histidine rich protein; hsp: heat shock protein; mdr: multi-drug resistance; Scfcu: Saccharomyces cerevisiae cytosine deaminase/uracil phosphoribosyl-transferase; Pbdt: Plasmodium berghei dhfr terminator; FRT: flippase recognition target; P: primer; TR: target region. Diagnostic long range (C) and intra-exonic (D) PCRs performed on genomic DNA from wild type (NF54, lane 1 or lane 3) and mutant (PfΔmdr2, lane 2 or PfΔmdr5, lane 4) lines, successfully confirmed deletion of Pfmdr2 and Pfmdr5 in which heterologous DNA was removed.