Enamel defects and ameloblast-specific expression in Enam knock-out/lacZ knock-in mice
Enamel defects and ameloblast-specific expression in Enam knock-out/lacZ knock-in mice
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DOI：
10.1074/jbc.m710565200
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发表时间：
2008-04-18
影响因子：
4.8
通讯作者：
Simmer, James P.
中科院分区：
文献类型：
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作者：
Hu, Jan C. -C.;Hu, Yuanyuan;Simmer, James P.
釉蛋白对正常牙釉质形成至关重要，人类釉蛋白基因的缺陷导致常染色体显性遗传的釉质发生障碍。我们使用基因靶向产生一个敲入小鼠携带无效等位基因的enamelin（Enam），有一个lacZ报告基因取代Enam翻译起始位点和基因序列，通过外显子7。通过Southern印迹和PCR分析证实了转基因的正确靶向。在Enam基因敲除的小鼠中，未检测到釉蛋白的表达。组织化学5-溴-4-氯-3-吲哚-β-D-吡喃半乳糖苷（X-gal）染色显示成釉细胞特异性表达釉蛋白。Enam（+/-）小鼠上颌切牙的牙釉质几乎正常，但下颌切牙变色，并且在与上颌切牙接触的地方倾向于快速磨损。Enam（+/-）小鼠没有显示真正的釉质。使用X射线照相术、微计算机断层扫描术以及光学和扫描电子显微镜记录Enam（+/-）小鼠牙釉质的变化，但未发现骨、牙本质或除牙釉质层外的任何其他组织的任何扰动。虽然一层厚厚的釉质蛋白覆盖着未萌出牙齿的正常牙本质，但von Kossa染色显示在这层蛋白质中几乎完全没有矿物质形成。然而，一个薄的，高度不规则的，矿化地壳覆盖的牙本质上爆发的牙齿，显然是由于形成和融合的小矿化灶（calcospherites）在更深的部分积累的釉质蛋白层。这些结果表明，釉蛋白的成釉细胞特异性表达，并揭示，釉蛋白是必要的适当的釉基质组织和矿化。
Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam(+/-) mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam(+/-) mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam(+/-) mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.