Fig. 3. Disruption of the PfRh2b and PfRh2a genes in 3D7 parasites. (A) The pHTkΔrh plasmid has the human dhfr gene (hdhfr) flanked by two target sequences for homologous recombination within the PfRh2a and PfRh2b genes. It also includes the thymidine kinase gene (Tk) for negative selection with ganciclovir. 3D7Δ2b1/2 and 3D7Δ2a1/2 are the cloned transfected lines showing the structure of the double recombination crossovers. The PfRh2a and PfRh2b genes are shown with relevant restriction enzyme sites: Sw, SwaI; S, SacI; A, AvaII; P, PvuII. The sizes are in kbp. (B) Southern blotting of genomic DNA from 3D7, 3D7Δ2a/b1 and 3D7Δ2a/b2 (uncloned population transfected with pHTkΔrh), 3D7/pHTk (uncycled parasites transfected with pHTkΔrh), 3D7Δ2b1 and 3D7Δ2b2 (cloned lines), 3D7Δ2a1 and 3D7Δ2a1/a2 (cloned lines). The left panel was digested with PstI and SwaI, whilst the other panels were digested with SacI, AvaII and PvuII. (C–F) PfRh2a and PfRh2b are not expressed in the parasites with the PfRh2a and PfRh2b genes, respectively, disrupted. Supernatants from 3D7Δ2a1, 3D7Δ2a2, 3D7Δ2b1, 3D7Δ2b2 and 3D7 were probed with the relevant antisera: (C) anti-PfRh2a sera; (D) anti-PfRh2b sera; (E) affinity-purified anti-PfRh2a/2b antibodies; (F) anti-MSP3 antibodies.