Fig. 2. Targeted disruption of Tshz3. (A) Gene targeting strategy. The mouse Tshz3 locus consists of two exons and one intron, and spans more than 75 kb; the first and second exons encode, respectively, 13 and 1068 amino acids. The in-frame insertion of the lacZ-coding sequence (hatched box) and the neomycin expression cassette (pTK-neo; black box) into exon 2 (ex2; light dotted box) resulted in complete deletion of the zinc-finger motifs and created an XbaI digest size difference between wild-type and targeted loci. Restriction sites are abbreviated as follows: B, BamHI; E, EcoRI; S, SalI, N, NotI, X, XbaI. (B) Southern blot analysis of DNA from wild-type (+/+) and targeted (+/-) ES cell clones using XbaI-digested genomic DNA and a 3' external probe (3' probe; white box in A). (C) Appropriate recombination 5' to the locus was confirmed by PCR with primers shown in A (short arrows). (D) PCR-based genotyping of wild-type (+/+), heterozygous (+/-) and homozygous null (-/-) embryos. (E) Immunostaining of TSHZ3 (left panel) on sections from E15.5 wild-type (+/+) and Tshz3^lacZ/lacZ mutant (-/-) ureter; TSHZ3 is not detected in mutant ureter. Co-immunostaining of TSHZ3 and β-gal (right panel) on a section from E14.5 Tshz3^lacZ/+ heterozygote ureter.