A point mutation resulting in an aspartic acid to alanine substition at residue 400 (D400A) was introduced at the endogenous locus by homologous recombination of a targeting vector also carrying an intronic neo cassette. The missense mutation was introduced into sequence encoding the proofreading domain. RT-PCR analysis of heterozygous ES cells showed comparable levels of wild-type and mutant transcript. Western blot analysis of fibroblasts derived from mutant mice further indicated normal levels of expression.