FIG. 1.

Targeted disruption of the CBF1 gene by deletion of exon 4. (a) Schematic view of the genomic structure of the CBF1 gene locus. Numbered bars represent the exons. A detailed view of the 9-kb EcoRI gene fragment encompassing exon 4 and flanking intron sequences (a, b, c, and d) used for construction of targeting constructs I, II, and III is shown as an insert. All three targeting constructs share a diphtheria toxin α expression cassette (DTA) and a dual positive selection cassette combining the neomycin (neo) or hygromycin (hyg) resistance gene with a fluorescent marker, EGFP or DsRed2, flanked by loxP sites. (b) Schematic overview of the NcoI fragments of the wild-type (F1) and the correctly targeted loci before (F2, F4, and F6) and after (F3 and F5) processing by Cre-recombinase detected by a 5′ external probe. Restriction enzyme sites: E, EcoRI; P, PpuMI; EN, EcoNI; A, ApaI; B, BsrGI; EV, EcoRV; K, KpnI; N, NcoI. (c) Southern blot of genomic DNA isolated from DG75 cellular clones, before and post targeting, digested with NcoI and hybridized to the 5′ external probe. The striped lines represent the sequences of the EcoRI fragment used for constructing the targeting vectors.