Generation of CAGp-IND-ephrin-B2 Tg mice.

(A) Representation of the Southern blot analysis of ES cell clone nos. 1-6. ES cell lines carrying a single copy of the CAGp-IND-ephrin-B2 vector fragment were established by Southern blot analysis ofXbaI-digested (upper) and BamHI-digested (lower) genomic DNA from each ES cell clone. Shown are restriction enzyme sites (B, BamHI; X, XbaI). Bars A and B indicate the location of probes for Southern blot analysis. Horizontal open arrowheads represent loxP sites. CAGp, CAG promoter; bsr, Blasticidin resistance gene; pA, polyadenylation signals. (B) To allow ephrin-B2 expression, selected cell clones were electroporated with the Cre expression vector, CAGp-Cre, and selected in G418. Representation of the Southern blot analysis ofXbaI-digested genomic DNA from the parental no. 2 clone (left) and its no. 2e subclone (right) in which the Blasticidin resistance gene is deleted by Cre-induced recombination. The expected fragment sizes are indicated in panels A and B. (C) X-gal staining of ES clone and its subclone shown in panel B. (D) Immunodetection of ephrin-B2 protein of ES clones (2 and subclone 2e) shown in panels B and C. Extracts (10 μg protein) from ES cells were used for Western blotting with an anti–ephrin-B2 antibody. (E) Southern blot analysis of XbaI-digested genomic DNA from F1 male mouse of lines no. 2 and no. 6 using probe A and B in panel A for analyzing the germline transmission. (F) Schematic representation of the experiment. To allow ephrin-B2 expression regulated by the Tie-2 promoter in vivo, CAGp-IND-ephrin-B2 Tg mice were crossed with Tie-2p-Cre mice.33 (G) X-gal staining of vessels in the skin of Tie-2p-ephrin-B2 Tg mice. Blue color indicates that ephrin-B2 is expressed in both arteries (A and arrowheads) and veins (V and arrows). (H) X-gal staining of the ascending aorta of Tie-2p-ephrin-B2 Tg mice. Arrows indicate that ephrin-B2 (blue color) was specifically expressed in endothelial cells. Bars indicate 50 μm.