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. 2010 Nov 30;5(11):e15121. doi: 10.1371/journal.pone.0015121

Figure 1. FLPe mediated excision of resistance markers from P.falciparum gene deletion mutants.

Figure 1

(A) Schematic representation of the genomic locus of wild-type (WT), gene deletion mutant Δp5236gfp before and after removal of the hdhfr::gfp resistance marker. The construct (pHHT-FRT-(GFP)-Pf5236) for targeting deletion of the p52 and p36 genes contains the two FRT sequences (red triangles) that are recognized by FLP. P1, P2: primer pairs for LR-PCR analysis; B (BclI),H (HindIII), E(EcoRI): restriction sites used for Southern analysis; cam: calmodulin; hrp: histidine rich protein; hsp: heatshock protein; fcu: cytosine deaminase/uracil phosphoribosyl-transferase; pbdt: P.berghei dhfr terminator. (B) Long range PCR analysis of genomic DNA from WT and mutants Δp5236 and Δp5236gfp before and after transfection with constructs containing FLP or FLPe, confirming removal of the hdhfr::gfp resistance marker in FLPe-transfected parasites. See A for location of the primers p1 and p2 and the expected product sizes (i.e. WT, 4.8 kb; Δp5236, 4.6 kb; Δp5236gfp, 5.2 kb; Δp5236gfp*FLPe and Δp5236*FLPe, 2.3 kb). (C) Southern analysis of restricted genomic DNA from WT and mutants before and after transfection with constructs containing FLPe, confirming removal of the hdhfr::gfp resistance marker in the FLPe-transfected Δp5236gfp mutant. Upper panel: DNA was digested with HindIII/EcoRI (probed with p52 targeting sequence); Lower panel DNA digested with BclI (probed with p36 targeting sequence). (D) Analysis of GFP expression in mutant Δp5236gfp before and after transfection with constructs containing FLPe, confirming removal of the hdhfr::gfp resistance marker in the FLPe-transfected parasites.