Generation of the cux/CDPΔHD hypomorphic allele.

(A) A targeting vector was designed to delete a 4-kb region encompassing the homeodomain within exons 20 and 21 and the intervening intron, with a PGK-neomycine resistance gene (neo), which provides a premature in-frame stop codon (*) for cux/CDP translation termination. Exons (black boxes) encoding cut repeat 3 (CR3, gray box) and the homeodomain (HD, open boxes) are shown above the wild-type gene. Primers for PCR detection of wild-type and targeted alleles with relative product sizes are shown as arrows below the schematics. The probe used for Southern detection and the fragments generated by digestion at the BamHI sites (letter “B”) are indicated. (B) Southern blot of BamHI-digested DNA from ES clones heterozygous (lanes 1 and 4) and homozygous (lanes 2 and 3) for the targeted allele. Southern blot was probed with a 4-kbBamHI/HindIII probe to detect the 8.5-kb wild-type and 4.5-kb targeted alleles. (C) Agarose gel demonstrating the products from multiplex PCR reactions to detect wild-type and knock-out alleles in wild type (+/+), heterozygous (+/ΔHD) and homozygous (ΔHD/ΔHD) mice. (D) Immunoblot of thymic nuclear extracts from wild- type mice (+/+), mice heterozygous for the ΔCR1 allele (+/ΔCR1), homozygous for the ΔCR1 allele (ΔCR1/ΔCR1), heterozygous for the ΔHD allele (+/ΔHD), and homozygous for the ΔHD allele (ΔHD/ΔHD), probed with anti-CDP antisera, demonstrates expression and size differences of the cux/CDPΔCR1 and cux/CDPΔHD proteins in comparison with wild-type cux/CDP. Molecular weight markers at 200 kd and 97.4 kd are shown.