Figure 3.

Generation of ECE-2–/– mice by homologous recombination. (a) Targeting strategy. The exon encoding the zinc-binding domain of ECE-2 is replaced with a neor cassette driven by the RNA polymerase II promoter. Two tandem repeats of thymidine kinase (TK) are used for the negative selection with FIAU (see Methods). PCR primers in the neor gene and 3′ region external to the short arm of targeting vector are shown by arrows. ^AExon numbers are indicated according to the equivalent exons of the human ECE-1 gene (32); other ECE-2 exons are not shown. (b) Southern blot analysis of tail DNA from the offspring of ECE-2+/– intercrosses. DNA was digested with BamHI and hybridized with a 3′ probe (indicated by the gray box in panel a). k.o., targeted allele; w.t., wild-type allele. (c) RT-PCR of the ECE-2 transcript using total RNA extracted from the brain of ECE-2+/+, ECE-2+/–, and ECE-2–/– mice. Primers amplify the exon encoding the essential zinc-binding domain of ECE-2. The upper panel shows that a 160-bp transcript fragment is detected in wild-type and ECE-2+/– mice, but is absent in ECE-2–/– mice. The middle panel shows that no transcripts are detected without RT incubation. The lower panel shows detection of β-actin transcripts, indicating intact RNA preparations in all lanes.