Figure 3. Stearic to oleic acid conversion in parasites overexpressing a Pfscd-gfp transgene.

(A) Schematic diagram of the cotransfected plasmids, the recipient cg6-attB locus and the recombinant locus produced upon integration of the Pfscd-gfp cassette (upper panel). The lower panel represents integration of Pfscd-gfp into the cg6-attB locus of 3D7^attB::Pfscd. Lane 1: 1 kb ladder. Lanes 2&4: integration of the blasticidin cassette into the locus was assayed by PCR using primers p1 and p2. Lanes 3&5: the presence of the complete Pfscd-gfp fusion was detected using primers p3 and p4. Lane 2&3: control 3D7^attB strain. Lane 4&5: 3D7^attB::Pfscd-gfp line. (B) 3D7^attB and 3D7^attB::Pfscd-gfp rings were labeled with 1 µCi of [¹⁴C]-stearic acid for 6 hrs at 37°C. Following extraction, equal counts (40,000 cpm) were loaded on a 10% silver nitrate-impregnated TLC plate. FAMEs were resolved using petroleum ether/diethyl ether (17/3, v/v) and revealed after overnight exposure to a Kodak Biomax MR film. The upper panel shows the fatty acid profile of two independent samples of each strain. Oleic acid methyl esters were scraped from the TLC plate and counted. Results are expressed as a fold increase of oleic acid production (normalized to a value of one in the 3D7^attB strain). The data in the lower panel represent the means of 4 experiments with standard deviations.