Targeted deletion of the mouse cubilin gene. A, is a diagram of the structural and functional domains of cubilin. Ligand binding regions indicated in the diagram are based on published studies [12, 33, 34]. B, is a diagram of the gene targeting strategy showing the organization of cubilin gene exons (vertical lines in uppermost model and rectangles in expanded region shown below), the wild-type and targeted alleles and the location of 5' and 3' DNA probes used for Southern blot analysis. White portions of boxed exons depict untranslated sequences. The promoter-less EGFP (containing Kozak and ATG sequences) and loxP-floxed neoR cassettes were inserted into exon 1, 33 bp upstream of the cubilin ATG and 16 bp downstream from the proximal-most transcription initiation site, resulting in a replacement of the majority of exon 1. Homologous recombination between the targeting construct and the cubilin locus results in deletion of exon 1 coding sequence and all of exons 2–6. C, Southern analysis of HindIII/HhaI and BglII digested DNA from representative wild-type (WT) and targeted (-/+) ES cell clones using 5' and 3' probes (left and right panels, respectively). The wild-type allele yields a 7 kb band and the knockout allele yields a 5.5 kb band when genomic DNA is digested with HindIII and HhaI and hybridized with the 5' probe. Additionally, the wild-type allele yields an 11.3 kb band and the knockout allele yields a 7 kb band when genomic DNA is digested with BglII and hybridized with the 3' probe. The results show that the correct recombinant allele is present in the selected ES cell clone. D, Southern analysis of BglII digested tail DNA from a wild-type (WT) and a correctly targeted heterozygous (-/+) mouse.