Two exons encoding amino acids 36-106 of the kinase domain were replaced with a PGK-neo cassette via homologous recombination. The targeted mutation destroys lyine 44, a residue necessary for kinase activity. Homozygous mutant embryos were detected by PCR genotype analysis up to day E14.5. Sequence analysis of RT-PCR products from mouse embryonic fibroblasts (derived from E12.2 homozygous mutant embryos) revealed an in-frame deletion from nucleotides 106-318. However, Western blot analysis using antibodies directed against the C-terminus of Ikbkb failed to detect protein product.