Figure 6.
Generation of ephrin-B1ΔPDZ mutant. (A) Wild-type ephrin cDNA (WT), or mutant cDNA (ΔPDZ and P-tyr) were transfected into 293T cells together with a flag-tagged PDZ domain. The EphB2-Fc recombinant protein was used to pull down ephrin and the amount of PDZ domain coimmunoprecipitated was evaluated with an anti-flag antibody. The right panel shows a quantification of the amount of PDZ domain associating with wild-type and mutant ephrin. The asterisk indicates a nonspecific band. (B, panel a) Wild-type efnb1 locus. (Panel b) Homologous recombination of the vector for the ΔPDZ mutation introduces a HindIII site. (C, panel a) Southern blot analysis of targeted ΔPDZ ES cells. Genomic DNA was digested with HindIII and the probe used is indicated in gray in panel b of part B. Genomic DNA from different ES clones was used as a template for PCR (panel b), using either mutation-specific primers (top) or primers specific to exon 5 (bottom). (D) ES cells treated with retinoic acid (RA) were analyzed by Western blot using the ephrin antibody (C18). Ephrin-B1 could be detected in wild-type ES cells (WT), ES cells targeted with the conditional allele of ephrin-B1 (lox), and ES cells targeted with ΔPDZ mutation (ΔPDZ). To ascertain that the signal was specific for ephrin-B1, we also tested ES cells deficient for ephrin-B1 (null). These cells were obtained by transiently expressing the Cre recombinase in ephrin-B1lox ES cells and screening for recombined clones. The asterisks indicate nonspecific bands. (E) Embryos obtained by injection of ES cells carrying the ephrin-B1ΔPDZ mutation into ROSA26 blastocysts were stained with X-gal to evaluate the degree of contribution of the mutant ES cells. Two different tissues (spinal chord, top and limb bud, bottom) from four different E14.5 embryos are presented (ch1–ch4). The embryos with the most contribution of the mutant ES cells (ch3 and ch4) presented a cleft palate (+), whereas the embryos with very little contribution of the mutant ES cells (ch1 and ch2) did not have a cleft palate (–). (F) Histological examination of E14.5 embryos obtained by injection of ES cells carrying the ephrin-B1ΔPDZ mutation into wild-type blastocysts. (Panels b,d) Palatal shelves (asterisks) were formed but failed to elevate and fuse in the chimeric embryo. (Panels a,c) Littermate without a cleft palate. Sections were stained with Alcian blue and Nuclear Fast red.