Supplementary Figure 7: Generation of conditional Crh knockout mice.

Strategy for targeted manipulation of the Crh locus. (a) Partial restriction maps of the wild-type locus and targeting vector. For better visualization, loxP, frt and attP sites are not to scale. (b) Recombined reporter allele (Crh^tz) following homologous recombination in embryonic stem (ES) cells. (c) Recombined floxed allele (Crh^flox) following removal of the frt-flanked reporter-selection cassette, and (d) conditional knockout allele (Crh^-) following Cre-mediated excision of the loxP-flanked exon 2. (e) Southern blot analysis of wild-type (+/+) and targeted ES cell clones (+/tZ). The external Crh 5’-probe was hybridized to EcoRI-digested genomic ES cell DNA. The targeted allele is indicated by the presence of an additional mutant (mt) 8.9 kb fragment. The Crh 3’-probe was hybridized to BamHI-digested DNA from the same ES cell clone confirming homologous recombination by detection of an additional mutant fragment at 8.3 kb. Homologous recombination was confirmed with two external probes in two independently prepared genomic DNA samples. (f) Functionality of the conditional Crh allele was confirmed by breeding Crh^flox mice to Cre-Deleter mice resulting in ubiquitous ablation of Crh mRNA expression throughout the brain determined by ISH (representative images from four independent experiments). (g) Quantification of Crh mRNA expression in (f), shown as percent change compared to littermate controls. Unpaired two-tailed t-test; OB (t₍₆₎ = 2.9, *p = 0.032); Pir (t₍₆₎ = 24.3, ***p < 0.0001); adBNST (t₍₆₎ = 5.6, **p = 0.0014); avBNST (t₍₆₎ = 10.7, ***p = 0.0013); CeA (t₍₆₎ = 5.7, **p = 0.0013); PVN (t₍₆₎ = 7.3, ***p = 0.0003); n = 4 mice/group, 1-2 sections/mouse). (h) Expectedly, ablation of CRH in Crh^-/- mice also resulted in strongly diminished hypothalamic-pituitary-adrenal axis activity evident by significantly reduced corticosterone levels measured in the morning (am), evening (pm), and following 10 min (stress) and 90 min (recovery) of restraint stress. Unpaired two-tailed t-test - am (t₍₂₀₎ = 2.6, p = *0.017, n = 11 mice/group); pm (t₍₂₀₎ = 4.1, ***p = 0.0005, n = 13 Crh^flox/flox, 9 Crh^-/-); stress (t₍₂₂₎ = 8.9, ***p < 0.0001, n = 13 Crh^flox/flox, 11 Crh^-/-); recovery (t₍₂₂₎ = 3.7, **p = 0.0013, n = 13 Crh^flox/flox, 11 Crh^-/-). Error bars represent s.e.m.