FIG 5 .

PfEH1 and PfEH2 can be knocked out in cultured parasites. (A and B) Southern blots of digested genomic DNA from the parent 3D7 line, vector, and double-knockout clones. Analysis at both the PfEH1 (A) and PfEH2 (B) loci demonstrate the simultaneous disruption of both genes in clones 3G and 11C, but not in clone 1E. The black arrows to the right of the blots indicate the expected size for correct integration, and the asterisk indicates a band of unknown origin. In panel B, the pUF-TK_PfEH2 vector [Vector (#)] was used instead of pCC1_PfEH2 (called pABH-DKO in reference 89). The schematic representations of the integration events are outlined in Fig. S2 in the supplemental material. (C) Growth of the 3D7 parent line and the two double-knockout (DKO) clones over 4 days. No differences were observed in growth between these three cell lines, with this graph representative of data from three independent experiments. The doubling times are 1.14 ± 0.12 days for the 3D7 parent, 1.16 ± 0.10 days for double-knockout clone 3G, and 1.26 ± 0.12 days for double-knockout clone 11C (not significantly different [P > 0.53] using pairwise t tests; n = 3 for each). The smooth curve is the fitted exponential growth equation. (D) Transcript levels of PfEH1 and PfEH2 in unsynchronized 3D7 laboratory strain compared to field isolates from malaria-infected pregnant women or malaria-infected children. The data shown were taken from Vignali et al. (48). (E) Normalized expression of PfEH1 or PfEH2 in two laboratory strains (Saint Louis STL-3D7 or MRA-3D7) and three laboratory-adapted field isolates from Cambodia (MRA-1237, MRA-1238, and MRA-1241). Quantitative RT-PCR was performed using probes against PfEH1 and PfEH2, with actin as a reference gene. Values are normalized to the average expression in STL-3D7 and MRA-3D7. Data were calculated from three independent RNA preparations (means plus SEMs [error bars]) and are not significantly different using a two-way ANOVA (P > 0.05).