A loxP site was inserted into intron 1 and an IRES2-DTR-EGFP-loxP-Neo-loxP cassette was inserted 114 bp downstream of the stop codon in the 3' UTR in exon 12. Correctly targeted ES cells were transiently transfected with cre recombinase and clones that had just the neomycin cassette removed were selected for mouse production. This left the human diphtheria toxin receptor gene (HBEGF)-EGFP fuson gene cassette in the 3' UTR. Very low levels of EGFP were detected in thymocytes. Thymocytes and T cells from homozygous mice expressed approximately 1.2-fold less LAT molecules than those of wild-type mice. T cell function was not impaired in these mice. Cre-recombinase expression will remove most of the coding sequence and the DTR/EGFP-expression cassette from the locus.