Fig. 1. Generation of splice site mutation to abolish Fgf8b expression in mouse. (A) Schematic representation of mutations in the Fgf8 locus. The change of sequences (bottom; mutations are shown in red) at the junction of intron (lowercase letters) and exon (capital letters) mutates the 5' alternative splice acceptor and creates an NdeI site (underlined). (B) Southern blot analysis to identify targeted embryonic stem (ES) cell clones. Asterisks indicate non-specific signals. (C) PCR and restriction digestion analysis to verify the point mutation. (D) Schematic representation of Fgf8 (top), Fgf8^b-neo (middle, left), Fgf8^b (middle, right) and Fgf8^neo (bottom) loci, and alterations in RNA splicing due to the point mutation and neo insertion. The Fgf8-neo hybrid transcript results from a cryptic splice donor and acceptor in the neo gene and in the intronic region 360 bp downstream to the neo gene, respectively. (E) Reverse transcriptase (RT)-PCR analysis of different Fgf8 splice variants in E7.5 embryos of indicated genotypes using primers 1 and 2 (shown in E). Fgf8 splice variants (a-h and unknown) are marked to the left. Notice that Fgf8b (asterisk) is missing in Fgf8^b-neo/b-neo and Fgf8^b/b embryos, whereas the neo insertion has no effect on the alternative splicing of the first four exons. (F) RT-PCR assay using primers 3 and 4 (indicated in D) reveals that the insertion of neo results in the production of Fgf8-neo hybrid transcripts. B, BamHI; E, EcoRI; X, XbaI.