The intron-exon junction sequence of exon 1D, taaagGTA was changed to catatGTA, abolishing the 5' alternative splice acceptor, while the downstream alternative splice acceptor in exon 1D remained intact. A neo cassette flanked with two loxP sites was placed 400 bp downstream of exon 1D. RT-PCR analyses revealed that Fgf8b and three minor b-type splice variants were absent in E7.5 mutant embryos. The insertion of neo produced an additional transcript resulted from a cryptic splice donor and acceptor in the neo gene and in the intronic region 360 bp downstream to the neo gene.