Figure 3. Schematic representation of the generation of FLPe-mediated ‘resistance marker-free’ P. falciparum mutants.

Standard gene deletion by double cross-over (DXO) homologous recombination (left hand side) is compared to gene deletion using the FLPe-recombinase method described in this paper (right hand side). Both methods are essentially identical up to 10 weeks. First transformed parasites are treated by on/off cycling with the antimalarial drug WR99210 (POS SELECTION) to select for mutant parasites where the plasmid has become integrated into the genome by single cross-over (SXO) homologous recombination. Next negative drug selection (NEG SELECTION) using the drug 5-FC is applied to select for those parasites where an internal recombination (DXO) between plasmid and genomic sequences has occurred and the target gene is deleted. At this stage all transformed parasites are GFP positive as the hdhfr-resistance marker is fused to GFP. At this point conventional DXO gene deletion parasites are cloned by a method of limiting dilution. At week 15 cloned parasites still containing the resistance marker (+SM; shown in the standard DXO genotype schematic as a green arrow) can be expanded. In the FLPe recombinase method the gene deletion mutants selected after positive/negative selection are not cloned but immediately transformed with a plasmid encoding the enhanced FLP recombinase (pMV-FLPe). This plasmid is maintained episomally through blasticidin selection (BSD SELECTION) for one week after which BSD selection is released and once these parasites are detected in culture they are cloned by limiting dilution. At week 18, only 3 weeks longer than standard method, these resistance marker–free parasites can be expanded. Removal of the resistance marker is confirmed by the absence of GFP-expression as recombination between the introduced FRT sites (red triangles) has occurred removing plasmid, gfp and drug resistance marker sequences (-SM).