Fig. 1. Generation of ES cells with targeted floxed E-cadherin intron 2. (A) Schematic representation of the E-cadherin locus (drawn to scale, 1). Exons are represented by vertical black bars, and nucleotide positions are given with respect to the transcription start site (+1). The locus was targeted with vector TV1 (2) and subsequently with TV2 (3), with detailed analysis after each step, finally resulting in the double-targeted allele (4) to delete intron 2 by Cre recombinase expression (5). For additional negative selection, a herpes simplex virus thymidine kinase gene (HSV-tk) was integrated in TV1 and betageo was fused in-frame to the E-cadherin start codon. In TV2 a hygromycin resistance cassette (hyg^r) under the control of the phosphoglycerol kinase promoter (PGK) was inserted in reverse orientation 5' of exon 3. Promoter (P), exons (E1, E2, etc.), loxP sites (red triangles), FRT sites (blue triangles), polyadenylation signals (striped boxes), transcription start sites (horizontal arrows), used restriction sites and probes (horizontal red bars) are given. The expected fragments of the Southern blot analysis for the homologous recombination of TV1 with probe a are indicated by green bars, and those for TV2 with probe f by blue bars. If both events occur at the same allele (in cis), a 46 kb fragment is expected after digestion with SalI and SgfI with probe e and with probe c (orange bar). (B) Southern blot analysis of BamHI-digested ES-cell DNA of gene targeting with TV1 as outlined in A. A 6.2 kb fragment was observed in wild-type clones (+/+), and an additional 9.2 kb fragment in recombined clones (+/lacZ). (C) Southern blot analysis of BamHI-digested ES-cell DNA of second gene targeting (TV2). Besides a 12 kb wild-type fragment, a 7 kb fragment was detected in successfully targeted clones (+/hyg). (D) Pulse-field electrophoresis separation of SalI/SgfI-digested ES-cell DNA of double-targeted clones analyzed by Southern blot, hybridized with probe e (left) or probe c (right). Events on the same allele are easily distinguishable by the appearance of a 46 kb fragment in both panels (arrowhead) in addition to the wild-type fragment (arrow). In clones with trans orientation, an additional fragment of >150 kb is visible with probe e (left, white arrow) and a different fragment of 90 kb with probe c (right, white arrow).