Fig. 2.

Characterization of the mice lacking brain AM. (A) Southern blot analysis of BamHI-digested ES cell DNA with the external 5′ probe (Left) and the internal 3′ probe (Right). The wild-type (w) allele renders a band of 17 kb whereas the mutant alleles (f) render 4- and 12-kb bands, respectively. (B) PCR analysis of tail DNA from 3-week-old mice with AMD primers (Table S1). After PCR, samples were digested with BamHI. The wild-type (w) allele produces a band of ≈400 bp whereas the mutant (f) is identified by a 200-bp band. Heterozygous mice (w/f) produce both bands. (C) Genotypes of offspring issued by the cross of 5 pairs of heterozygous mice for the ubiquitous deletion. No homozygous mutant mice (−/−) were produced. (D) Real-time PCR quantification of the levels of AM produced in the brain and heart of wild type (wt/wt Cre+) and brain conditional AM mutants (f/f Cre+). While a large diminution is appreciated in the mutant brain, the heart AM contents do not show any significant difference with the wild type. (E) Real-time PCR quantification of the levels of AM and AM-related molecules CLR, RAMPs, AM2, and CGRP in the brain of wild-type and mutant animals. The drastic reduction of AM in the mutant brain does not affect the expression of the related genes.