Fig. 1. Generation of mice with systemic NF-B suppression. (A) Integration of IBN (Krappmann et al., 1996) into the ß-catenin locus by homologous recombination. IBN lacks the phosphorylation and ubiquitination sites, which are required for signal-induced degradation (Krappmann et al., 1996). The murine ß-catenin locus (exons, white boxes) and targeting vectors are shown. IBN and loxPIBN cDNAs were inserted in frame to the start codon in the second exon of the ß-catenin gene and replaced exons 3 to 6, resulting in a ß-catenin null allele (Huelsken et al., 2000). In c^loxPIBN, a stop codon inserted in frame is flanked by loxP sites (black arrowheads) (Zhang et al., 1996). (B) Western blot of c^loxPIBN (D5-C7) and c^IBN (D7-H9) ES cell clones using an antibody directed against the IB C-terminus (C-21). ns, non-specific. IBN protein is detected only in the ES cells carrying the IBN transgene. (C) EMSA of c^IBN ES clones (G2 and G3), and c^loxPIBN clones (B7 and A3) before Cre-mediated recombination as controls. ES clones were treated with PMA and specific complexes inhibited with anti-p65 antibody, as indicated. NF-B activity can no longer be induced by PMA in c^IBN ES cells clones. (D) Embryonic fibroblasts (MEFs) of wild-type (WT) and c^IBN littermates (IBN) were stimulated with IL1ß and TNF for the times indicated and extracts were assayed by EMSA. In c^IBN fibroblasts, DNA-binding activity of NF-B p50/p65 complexes is severely blocked after stimulation with TNF and completely impaired after IL1ß stimulation. (E) The same extracts were analyzed in Western blots for IB and ß-catenin proteins, as indicated. As expected, endogenous IB is degraded after stimulation leading to the observed variations of the protein in wild-type and c^IBN fibroblasts. De novo synthesis of IB protein, which depends on active nuclear NF-B complexes, is delayed in c^IBN fibroblasts, as NF-B translocation is strongly suppressed. (F) A wild-type littermate compared with a c^IBN mouse (right). (G) Increased apoptosis in the embryonic liver of c^IBN mice. Cryosections of embryonic livers of wild-type and c^IBN embryos at E12.5, E14.5 and at birth, P0, as indicated, were analyzed by in situ TUNEL assay.