FIG. 1.

Construction of the KNT targeting vector and generation of mutant mice. (A) Schematic representation of the genomic knt locus, the targeting construct (kntTV) and the different mutated alleles. Homologous recombination of kntTV introduces a loxP-flanked neomycin resistance gene (neo) into intron 2 and a single loxP motif into intron 5 of the knt genomic locus. Cre-mediated recombination at the targeted locus (T) produces either a floxed knt allele (F) or a deleted knt allele (Δ) by excision of the neomycin resistance gene or by deletion of the neomycin resistance gene together with exons 3 to 5, respectively. Exons are depicted as shaded boxes; loxP motifs are indicated as filled triangles. Transcriptional directions of neomycin resistance and tk (open boxes) are indicated by arrows. fl3, 3′ external probe; neo1, neo probe; E, EcoRV; P, PstI; C, ClaI; S, SacI; N, NcoI; K, KpnI; M, MfeI; M/EI, MfeI/EcoRI. (B) Southern blot analysis of genomic DNA from targeted ES cell clones before and after in vitro Cre recombination (left) and from mouse tail biopsies (right). A 1.8-kb restriction fragment after PstI digestion is diagnostic for the mutated alleles in contrast to a 2.6-kb fragment generated from the wild-type allele. Note that the deleted allele is cut at an endogenous PstI site and not at the PstI site introduced with the distal loxP motif. EcoRV digestion generates fragments of 8.5 kb (Δ), 11.2 kb (T), and 17.7 kb (wt and F) in size. Southern detection of the PstI and the EcoRV digests was performed with the external probe fl3. Hybridization with neo1 after ClaI/SacI double digestion yields one 12-kb fragment for the targeted allele (T), indicating a single integration event. E14, genomic DNA from E14.1 ES cells.