Figure 3

Creation of two PF11_0394/GFP clonal parasite populations for protein trafficking studies. A) Transfection schematic demonstrating how the PF11_0394/GFP construct was created using the pPM2GT vector (obtained from MR4) and successfully incorporated into the genome of the parasite via homologous recombination technology [19]. Expression of GFP is driven by the endogenous promoter of PF11_0394. Generation of two PF11_0394/GFP clonal parasite populations were verified by both PCR analysis (B) and Southern blot analysis using digoxigenin (DIG) technology coupled with autoradiography (C). Arrows indicate the predicted integration products of 10,454 base pairs and 6,602 base pairs using a PF11_0394 specific probe after restriction digestion with SapI and KpnI. The product indicated by an asterisk is of unknown origin and is likely a rearrangement of the plasmid that occurred after transfection. The drug cassette within the vector used for positive selection is human dihydrofolate reductase (hDHFR). GFP = Green fluorescent protein, Wt = Wild-type parasite genomic DNA, Cl = Clonal PF11_0494/GFP parasite genomic DNA, and Ep = PF11_0394/GFP plasmid DNA representing the episome. The arrows on the transfection schematic represent primers used for PCR analysis.