Generation of End^–/– mice. (a) Targeting strategy. Homologous recombination deletes 609 bp of the End gene including exon 1 (E1; 66 bp), placing the LacZ gene under control of its promoter. The targeting construct contains a 1.4-kb SmaI fragment (5′ HR) and a 5.1-kb SmaI-BglII fragment (3′ HR) of the End gene flanking the LacZ-Neo cassette. Proper recombination events were screened by Southern blot analysis with 5′ external and LacZ internal probes. Bm, BamHI; Bg, BglII; EI, EcoRI; Sa, SacI; Sc, ScaI; Sm, SmaI; Xb, XbaI. (b) Identification of targeted ES cell lines. DNA from 2 geneticin- and ganciclovir-resistant targeted ES cells (4A-11, 4A-36) and a wild-type clone (WT) were digested with ScaI. The 5.4-kb WT and 7.3-kb recombinant alleles hybridizing with the 5′ external probe and the recombinant allele reacting with the LacZ probe are shown. (c) Genotyping of embryos by multiplex PCR. DNA from yolk sacs of an E9.5 litter derived from a C57BL/6 End^+/– mating is shown; #21 represents the mother and C represents the PCR control reaction without DNA. Primers ME1F and ME1R amplify normal exon 1 (300 bp), ME2F and ME2R amplify normal exon 2 (383 bp), and ME1F and MEZR specifically amplify the recombinant product (476 bp). (d) Absence of endoglin mRNA in End^–/– embryos. RNA was prepared from the same embryos as in c and analyzed for endoglin and β-actin mRNA by RT-PCR.