Targeted disruption of the PfMSP-8 gene in P. falciparum line D10. (A) Schematic diagram outlining the strategy used to target the endogenous PfMSP-8 gene and generate the parasite lines D10-MSP-8 3′ and D10-ΔMSP-8. The sequences encoding the signal peptide and the EGF-like domains are indicated by the black and grey boxes, respectively. hDHFR, human dihydrofolate reductase. (B) Southern blot of restricted genomic DNA isolated from the parental P. falciparum D10, D10-MSP-8 3′, and D10-ΔMSP-8 parasites. The blot was probed with a PfMSP-8 targeting sequence (solid line in panel A). (C) Western blots of protein extracts from mixed schizont- and ring-stage D10-MSP-8 3′ (WT) and D10-ΔMSP-8 (Δ8) parasites probed with mouse monoclonal antibody 4H9/19 specific for PfMSP-119 (αPfMSP-1) (left panel) and rabbit antisera specific for the EGF-like domains of PfMSP-8 (αPfMSP-8) (right panel). The locations of full-length 200-kDa MSP-1 and the processed 42-kDa and 19-kDa forms of PfMSP-1 are indicated on the left, while the positions of the 80-kDa and 17-kDa PfMSP-8 species are indicated on the right. (D) Western blots of protein extracts from several times throughout the erythrocytic life cycle of D10-MSP-8 3′ and D10-ΔMSP-8 parasites. The numbers at the top indicated the times (in hours) after erythrocyte invasion, and the letters indicate the erythrocytic life cycle stages, as follows: R, ring stage; T, trophozoite stage; S, schizont stage. Identical blots were probed with a polyclonal rabbit antiserum specific for the EGF-like domains of PfMSP-8 (αPfMSP-8) and polyclonal rabbit antisera specific for the PfHSP-70 protein (αPfHSP-70).