Fig. 1.

Gene targeting of the ACP locus using a positive/negative selection scheme to enrich homologous recombinants. (A) Schematic representation of the two-marker targeting plasmid and positive/negative selection for homologous recombination at the ACP locus. (B) FACS profiles of the nonfluorescent wild type (RH, short-dashed line), a YFP-expressing clone derived from this strain (long-dashed line), and a population of stable drug-resistant parasites after transfection with the double-marker targeting plasmid (solid line, putative KOs; the indicated 1% least fluorescent of parasites were sorted and cloned). Two of 25 of these clones showed successful targeting of the locus (compared with >400 clones unsuccessfully screened by using a single-marker approach). (C) Allelic replacement of the native ACP gene by chloramphenicol acetyl transferase (CAT) gene yields altered restriction profile after digestion with restriction enzymes BamHI, BglII, and XhoI. (D) PCR detection of endogenous (1.5 kb) and inducible (1.3 kb) ACP genes. Heterolog., clone harboring a heterologous insertion of the targeting plasmid in addition to the native locus. The pKO targeting plasmid and RH genomic DNA serve as control for KO insert and native ACP, respectively. (E) Southern blot analysis of BamHI/BglII and BamHI/XhoI digests of RH and ΔACP/ACPi genomic DNA with probes P1 (ACP intron, hybridizes to native ACP but not to the ectopic minigene copy), P2 (CAT coding sequence), and P3 (3′ noncoding region present in both loci). All autoradiographs show the same membrane, which was stripped and reprobed.