Fig. 1. Generation of the Floxed ephrinB2 locus. (A) Restriction maps of the wild-type ephrinB2 locus, the targeting vector, the initial targeted locus, floxed locus after neo deletion and targeted locus after complete deletion. The targeting vector contains loxP sites (arrowheads) flanking the first coding exon (gray bar). It also contains a floxed PGK-neomycin (‘PGKneo’) selection cassette that was subsequently removed by transient Cre expression to avoid disturbing normal ephrinB2 transcription (see C). (B) Confirmation of homologous recombination of the targeting vector by Southern blotting. The ES cell genomic DNA has been digested with HindIII, and hybridized with Southern probe A (see A). Wild-type (6 kb) and targeted (4 kb) loci differ by a HindIII site flanking the 5' loxP site (see A). (C) Identification of ES cells that have undergone PGKneo cassette deletion (see A) after transient Cre recombinase expression. Genomic DNA was digested with HindIII, and hybridized with southern probe B (see A). ‘Neo deleted’ indicates loss of PGKneo cassette with retention of the first exon. Deletion of the entire region or deletion of the floxed exon are not distinguishable from wild type in this Southern blot. (D) Confirmation of ephrinB2 exon 1 deletion in mice. Progeny of an ephrinB2^loxP/+ X CMV-Cre cross show intact (long) or deleted (short) PCR products with primers specific for the loxP allele.

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