Figure 2

Generation of drug-selectable marker-free Δp230p-GFP and Δp230p-GFP-LUC P. berghei ANKA parasites.

(A–B). Replacement strategy to generate Δp230p-GFP (A) and Δp230p-GFP-LUC (B) parasites. The wild-type (WT) genomic locus of P. bergheiP230p was targeted with GOMO-GFP (A) and GOMO-GFP-LUC (B) replacement plasmids containing a 5′ and a 3′ homologous sequence inserted on each side of the plasmid cassettes. Upon a double crossover event, the P230p gene is replaced by the GFP(-LUC)/hDHFR-yFCU/mCherry triple cassette. Recombination between the two identical PbDHFR/TS 3′ UTR sequences (pink lollipops) results in excision of hDHFR-yFCU and mCherry. Genotyping primers and expected PCR fragments are indicated by arrows and lines, respectively. The Southern probe and expected restriction fragments are also shown. (C–D). FACS scatter plots of parasites transfected with GOMO-GFP (C) and GOMO-GFP-LUC (D) constructs targeting PbP230p. GFP⁺ mCherry⁺ parasites obtained after positive selection with pyrimethamine (left panels) are replaced by GFP⁺ mCherry⁻ parasites after negative selection with 5-FC (right panels). (E). Southern blot analysis of genomic DNA isolated from P. berghei WT, Δp230p-GFP and Δp230p-GFP-LUC parasites after positive selection with pyrimethamine (1^st) or negative selection by 5-FC followed by FACS sorting of GFP⁺ mCherry⁻ parasites (final), using a probe specific for the 3′ homology sequence of PbP230p. After digest with NheI and AflII, the probe hybridizes to fragments of 8.3 kb in WT, 5.5 kb in recombined non-excised parasites and 5.0 or 7.3 kb after marker excision in the final Δp230p-GFP and Δp230p-GFP-LUC parasite populations, respectively. (F–G). PCR analysis of genomic DNA isolated from P. berghei WT, Δp230p-GFP (F) and Δp230p-GFP-LUC (G) parasites recovered after the first positive selection with pyrimethamine (left panels), after the second negative selection with 5-FC (middle panels) and after flow cytometry sorting of GFP⁺ mCherry⁻ parasites (final populations, right panels). Confirmation of the predicted recombination events was achieved with primer combinations specific for 5′ or 3′ integration. An additional primer combination was used to assess removal of the hDHFR-yFCU selectable marker (3′ excised). A wild type-specific PCR reaction (WT) confirmed the absence of residual wild-type parasites in the final GFP⁺ mCherry⁻ FACS-sorted parasites.