Figure 5

A-C. Generation of PF3D7_1363700-GFP clonal populations that appear to express PF3D7_1363700-GFP during the intra-erythrocytic stages. A stable integration event into the PF3D7_1363700 chromosomal locus was detected via genotype analyses. (A) For PCR analysis, a genomic DNA template from wild-type parasites (WT) was used as a control and MpPM2GT transfected culture DNA before limiting dilution (Preclonal) was used to demonstrate the presence of integrants and wild-type parasites carrying the episome. Clonal population MpPM2GT-81 (81) was the experimental template. PCR primer combinations listed to the left of the figure were used to detect: 1) wild-type parasites: gene specific forward and reverse (GSPF/R); 2) episomes: hDHFR F/R and 3) parasites with integration: gene specific forward (GSPF) and GFP reverse (GFPR). The faint band that represents the detection of episome in wild-type control DNA was due to primers binding to DNA at a temperature lower than the ideal annealing temperature. At ideal annealing temperatures, this band is not present. (B) For Southern blot analysis, integration was detected by the presence of a 4.2-kb band while wild-type parasites with episome were represented by 2.7-kb and 5.1-kb bands. The asterisk denotes an unknown band, which could be due to plasmid rearrangement after transfection. Genomic DNA from wild-type P. falciparum NF54 parasites (WT) and transfected episome (P) were used as controls in the Southern blot experiments and a digoxigenin-labelled PF3D7_1363700 DNA probe was used for detection. (C) PF3D7_1363700-GFP fusion protein (MGFP) from clonal population 81 appears to be present using anti-GFP antibodies for detection as denoted by the arrow. Wild-type (WT) and 3D7HT-GFP (GFP+) parasites were used as negative and positive controls, respectively, in the Western blot.