FIG. 1.
Targeted disruption of the KAP2/KAP3 locus of C. fasciculata. (A) Schematic representations of the KAP2 and KAP3 genomic locus, targeting vectors, and targeted alleles. Diagrams indicate the size differences between the wild type, the kap2/3+/− single-disrupted alleles, and the kap2/3−/− double-disrupted alleles and show the positions of XhoI restriction sites (X). The gene-targeting vectors contain the BSD or HYG selectable markers flanked by the L. major 5′ and 3′ flanking regions of the DHFR-TS gene for expression of the drug resistance markers. UTR, untranslated region. (B) Southern blot of XhoI digests of C. fasciculata DNA from wild-type cells and from kap2/3+/− cells in which one allele of KAP2 and KAP3 was disrupted by insertion of the BSD cassette or kap2/3−/− cells in which the remaining alleles were disrupted by insertion of the hygromycin cassette (BSD/HYG). (C) Absence of KAP2 and KAP3 proteins in the kap2/3−/− double-mutant cells. Cell lysates of 2 × 106 wild-type, kap2/3+/− single-mutant, and kap2/3−/− double-mutant cells were analyzed by Western blotting with either affinity-purified KAP2 or KAP3 antibodies.