Fig. 3. Generation of Nuf2 and Hec1 clones carrying transgenes under the control of a tet-repressible promoter. (A) Restriction maps of the Nuf2 and Hec1 loci, gene disruption constructs, and targeted loci. Black boxes indicate the positions of exons. Several restriction enzyme sites are shown. The position of the probe used for Southern hybridization is indicated. (B) Restriction analysis of targeted integration of the Nuf2 and Hec1 disruption constructs. In Nuf2 disruption, genomic DNAs from wild-type DT40 cells (Wt), a clone after first round targeting (+/–, 1^st), a clone after first round targeting and random integration of the Nuf2 transgene (+/–Nuf2+, 1^st +cDNA), and a clone after second round targeting (–/–Nuf2+, 2^nd +cDNA) were analyzed by Southern hybridization with the probe indicated in (A). In Hec1 disruption, genomic DNAs from wild-type DT40 cells (Wt), a clone after first round targeting (+/+/–, 1^st), a clone after second round targeting (+/–/–, 2^nd), a clone after second round targeting and random integration of the Hec1 transgene (+/–/–Hec1+, 2^nd+cDNA), and a clone after third round targeting (–/–/–Hec1+, 3^rd+cDNA) were analyzed by Southern hybridization with the probe indicated in A. (C) Western blot analysis of whole cell extracts of Nuf23-63 and Hec25-1 cells with anti-Nuf2 and anti-Hec1 antibodies at the indicated times following addition of tet. Equal amounts of extracts were separated by SDS-PAGE and analyzed by western blotting. Anti--tubulin antibody was used as a loading control.