Figure 3

Generation of drug-selectable marker-free Δslarp-GFP and Δslarp-GFP-LUC P. berghei ANKA parasites.

(A–B). FACS scatter plots of parasites transfected with GOMO-GFP (A) and GOMO-GFP-LUC (B) constructs targeting PbSLARP (PBANKA_090210). The GFP⁺ mCherry⁺ parasite population obtained after positive selection with pyrimethamine (left panels) was replaced by a GFP⁺ mCherry⁻ population after negative selection with 5-FC (right panels). (C). PCR analysis of genomic DNA isolated from P. berghei WT, Δslarp-GFP (left panel) and Δslarp-GFP-LUC (right panel) parasites recovered after GOMO selection. Confirmation of the predicted recombination events was achieved with primer combinations specific for 5′ integration (5′ integr.) or 3′ integration followed by marker excision (3′ excised). Primers used for genotyping are indicated in Fig. S6 and Table S1. The absence of amplification with primer combinations specific for the WT locus (WT) and the non-excised integrated construct (3′ integration) confirmed that the final populations contained only Δslarp marker-free parasites. (D). Southern blot analysis of genomic DNA isolated from P. berghei WT, Δslarp-GFP and Δslarp-GFP-LUC final parasite populations selected with the GOMO procedure, using a digoxigenin-labelled probe specific for the 3′ homology sequence of PbSLARP. After digest with SnaBI and AflII, the probe hybridizes to a 11.6 kb fragment in WT, a 4.0 kb fragment in recombined non-excised parasites and a 3.6 or 5.8 kb fragment after marker excision in the final Δslarp-GFP and Δslarp-GFP-LUC parasite populations, respectively. The position of the probe and the restriction fragments are indicated in Fig. S6.