The coding sequence for wild-type Hesx1 was mutated by PCR so that codon 26 (isoleucine) was changed to encode a threonine residue (ATT->ACC). A loxP-flanked PGK-Neo selection cassette was inserted into the first intron of the mutated Hesx1 sequence in the targeting vector. Homologous recombination in CCE ES cells replaced the wild-type sequence with the mutant sequence in the targeting vector. F1 animals were crossed with Actin-beta-cre mice to excise the PGK-Neo cassette. (J:142649)