Fig. 7.

Production of transgenic P. falciparum lines expressing chimeric MSP1.A. PCR analysis to detect correct genomic integration of the homologous recombination constructs pMSP1chimWT and pMSP1chimSUB2to1. As controls, template DNA was prepared from the parental 3D7 clone, or input pMSP1chimWT plasmid DNA only (P). For test samples, genomic DNA was prepared after four rounds of drug cycling from three lines transfected with pMSP1chimWT (a–c), or three lines transfected with pMSP1chimSUB2to1 (d–f), or from one of the pMSP1chimSUB2to1-transfected line following seven drug cycles (d*). Oligonucleotide primers used in the PCR were designed to hybridize upstream of the region used as the targeting sequence in the 3D7 MSP1 locus, as well as to an allele-specific portion of the recodonized Wellcome sMSP1-19+ sequence in the transfection constructs (see Fig. 6C). A PCR product of approximately 1.2 kb can therefore only be produced if the predicted integration event has taken place. The integration-specific PCR product was detected for all three pMSP1chimWT-transfected lines but not for the pMSP1chimSUB2to1-transfected lines, except for one line examined after seven drug cycles (d*) (arrowed).B. IFA examination of the uncloned transgenic lines confirms expression of chimeric forms of MSP1. Samples of the pMSP1chimWT-transfected line (line a, drug cycle 4) and the pMSP1chimSUB2to1-transfected line (line d*, drug cycle 8) were double-probed with the 3D7 MSP1-specific mAb X509 and the Wellcome MSP1-specific mAb 111.4. Whereas the parental 3D7 and T9/94 parasite lines showed reactivity only with one or other type-specific mAb as expected, both transgenic populations contained parasites reactive with both mAbs, indicating expression of the predicted chimeric MSP1. The proportion of schizonts in the transgenic lines reactive with both antibodies was approximately 95% for the pMSP1chimWT-transfected line, and approximately 0.2% for the pMSP1chimSUB2to1-transfected line. Nuclei were counterstained with DAPI. Scale bar, 5 µm.