Fig. 2. Generation of Tcf3^-/- mice. (A) Targeting strategy. Exons 1-3 of Tcf3 are shown as boxes along the unbroken line representing the Tcf3 gene fragment present in the targeting construct. Broken lines represent Tcf3 gene fragments flanking the targeting construct. The numbers above double-sided arrows represent the size in kb of the fragments generated by digestion with either Bgl2 (B) or HinCII (H). The triangles between exons 1 and 2 and flanking the PGK-NEO cassette represent loxP sites for Cre-mediated recombination. Small single-sided arrows depict the position of forward (for) and reverse (rev) PCR primers used for genotyping of mice. (B) Homologous recombination events in ES clones were confirmed by Southern blotting. +/+, ES cells prior to electroporation; +/neo, one ES clone that had correctly integrated the targeting vector fragment into the Tcf3 locus; +/-, a clone derived from the +/neo clone after electroporation with Cre-recombinase was used to remove the PGKNEO cassette and exon 2 of Tcf3. (C) Genotyping reaction with primers shown in A shows the loss of one copy of exon 2 in Tcf3^+/- ES cells and mouse embryos, and the loss of both copies of exon 2 in Tcf3^-/- embryos. (D) Western blot of total protein from E8.5 embryos with an antibody directed towards the C terminus of Tcf3 protein confirms loss of Tcf3 protein in Tcf3^-/- embryos.