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Generation of HB-EGF KO mice.

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posted on 14.10.2009, 02:44 by Atsushi Oyagi, Yasuhisa Oida, Kenichi Kakefuda, Masamitsu Shimazawa, Norifumi Shioda, Shigeki Moriguchi, Kiyoyuki Kitaichi, Daisuke Nanba, Kazumasa Yamaguchi, Yasuhide Furuta, Kohji Fukunaga, Shigeki Higashiyama, Hideaki Hara

(a) Gene-targeting strategy. Mouse Hb-egf cDNA containing the polyadenylation (pA) sequence flanked by loxP sequences was fused with the first exon of the mouse Hb-egf gene. The lacZ gene was inserted downstream of the Hb-egf cDNA. The targeting vector also contains the neomycin-resistance gene (neo), driven by the phosphoglycerate kinase (PGK) promoter and the diphtheria toxinA-fragment (DT-A) gene. Cre-mediated recombination results in the deletion of Hb-egf cDNA and in the expression of the lacZ gene. Exon sequences are indicated as black boxes. E, EcoR1; H, hind1; K, Kpnl; S, Sacll; V, EcoRV; X, Xhol. (b) Genotype of ventral forebrain-specific HB-EGF KO mice. Ventral forebrain-specific HB-EGF-deficient mice were confirmed by PCR as lox homozygous and Cre-recombinase positive. (c) Coronal section through the cortex region; square indicates area shown in the photomicrographs. (d) Histological analysis of cortex from controls (upper) and HB-EGF KO (lower) adult mice. LacZ staining, scale bar = 50 µm. In situ hybridization using an Hb-egf probe, scale bar = 100 µm. Immunohistochemical staining with anti-HB-EGF antibody, scale bar = 20 µm. Cresyl violet staining, scale bar = 500 µm. (e) Coronal section through the hippocampus; square indicates area shown in the photomicrographs (CA1, CA3, and DG). (f) LacZ staining of whole and individual hippocampal region in HB-EGF KO mice, scale bar = 500 µm. (g) Immunohistochemical analysis of individual hippocampal region from controls (upper) and HB-EGF KO (lower) adult mice, scale bar = 20 µm.

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