Generation ofcriptoF78A/F78Amutant mice by homologous recombination in ES cells. (A) Strategy for inserting the F78A substitution at the cripto locus. Numbered boxes represent wild-type exons (black) and the mutant exon carrying the F78A substitution (gray). Location of PCR primers (black arrowheads) and probes (black bars) used for genotyping are indicated. B, BamHI; E, EcoRV; H HindIII; P, PstI. (B) Genotyping of cell lines by Southern blot analysis. Genomic DNA from wild-type (+/+) and floxed (+/F78A) ES cells was digested with EcoRV and hybridized with the 5′ (RH5) and the 3′ (BE6) probes shown in A. The sizes of hybridized fragments are indicated in kilobases. (C) Genotypes of offspring from cripto+/F78A heterozygote intercrosses. Primers c and d amplified a 280-bp fragment of the wild-type allele and a 400-bp fragment of the Cre-deleted allele. (D) Spatial distribution of both wild-type (left) and F78A mutant (right) Cripto protein. Wild-type and criptoF78A/F78A embryos were immunostained with anti-Cripto antibodies at 6.75 dpc. Bars, 50 μm.
