Development and Validation of Novel Amelogenesis Models

摘要 这项研究建议集中在釉质发生的分泌阶段，在那里釉质矿物带开始 在牙本质矿物上，并拉长，直到牙釉质层达到其最终尺寸。许多基因导致遗传 釉质畸形(釉质发育不全，AI)。分泌期必需的基因缺陷 通常会导致牙釉质变薄(发育不良)，表面粗糙。牙釉质在特定的细胞外形成 由成釉细胞建立和修改的空间。我们引用了19个导致釉质发育不全的人类AI基因， 但重点放在那些与细胞外釉质基质最直接相关的物质上。五个基因编码分泌 基质蛋白：ENAM、AMBN、AMELX、MMP20和ODAPH。除了ODAPH(以前的C4orf26)，这些 基因/蛋白质都有很好的特性。有两个关键的分泌期离子转运蛋白：SLC4A4(NCBe1； 将重碳酸盐输送到牙釉质基质中)和SLC13A5。众所周知，重碳酸盐可以中和产生的酸。 但为什么SLC13A5编码的柠檬酸转运蛋白(NACT)的缺失会导致严重的 釉质畸形是未知的。 SLC13A5在软(肝、脑)和硬(骨、牙)组织中是必需的。令人惊讶的是~80%的柠檬酸盐 身体是骨子里的。柠檬酸是柠檬酸循环的一部分，在柠檬酸循环中，柠檬酸是由草酰乙酸酯、乙酰基- 辅酶A和水在线粒体基质中由柠檬酸合成酶催化的反应中。可能是柠檬酸盐 扮演两种角色之一：它可以通过成釉细胞近端膜转运到成釉细胞(流入)以增加 能量代谢或从成釉细胞外(外排)穿过其远端膜进入发育中 釉质基质。根据骨骼和牙釉质中柠檬酸盐的发现，我们假设柠檬酸盐是分泌的，并有助于 以调节釉质带的沉积。ODAPH和SLC13A5在釉质带形成中的关键作用 代表了我们对釉质发生的理解中的两个主要差距。我们通过生产和生产来缩小这些差距 Odaph和Slc13a5基因敲除(KO)小鼠表达人类同源早停密码子的验证 导致人工智能的突变。我们还产生了一个Slc13a5敲打蛋白(Ki)，在其C末端表达3个标志表位。 提出了两个具体目标： SA1：建立和验证与人类AI同源的Slc13a5 KO小鼠模型(p.Arg333*)，并 制备一只SLC13a5FLAG Ki小鼠，用于敏感和特异的免疫定位(IHC)。 UG3：使用CRISPR/Cas9在C57BL/6J背景下生成Slc13a5/-小鼠AI模型。 UG3：建立SLC13a5FLAG小鼠野生型标记模型，用于敏感和特异的定位。 UH3：通过鉴定牙釉质来验证Slc13a5-/-小鼠。用免疫组织化学方法验证Slc13a5FLAG小鼠。 SA2：建立和验证与人类AI同源的Odaph KO小鼠模型(p.Cys43*)。 UG3：使用CRISPR/Cas9在C57BL/6J背景下生成Odaph-/-小鼠AI模型。 UH3：通过鉴定Odaph-/-小鼠的釉质表型来验证其有效性。

Abstract This research proposal focuses on the secretory stage of amelogenesis where enamel mineral ribbons initiate on dentin mineral and elongate until the enamel layer reaches its final dimensions. Many genes cause inherited enamel malformations (amelogenesis imperfecta, AI). Defects in genes necessary for the secretory stage typically cause thinner (hypoplastic) enamel with a rough surface. Dental enamel forms in a defined extracellular space that is established and modified by ameloblasts. We cite 19 human AI genes causing enamel hypoplasia, but focus on those most directly associated with the extracellular enamel matrix. Five genes encode secreted matrix proteins: ENAM, AMBN, AMELX, MMP20, and ODAPH. Except for ODAPH (formerly C4orf26), these genes/proteins are well-characterized. There are 2 key secretory stage ion transporters: SLC4A4 (NCBe1; transports bicarbonate into enamel matrix) and SLC13A5. Bicarbonate is known to neutralize the acid generated by mineral deposition, but why absence of the citrate transporter (NaCT) encoded by SLC13A5 causes severe enamel malformations is unknown. SLC13A5 is required in soft (liver, brain) and hard (bone, teeth) tissues. Surprisingly ~80% of all citrate in the body is in bone. Citrate is part of the Citric Acid Cycle where citrate is generated from oxaloacetate, acetyl- coenzyme A, and water in a reaction catalyzed by citrate synthase within the mitochondrial matrix. Citrate likely plays 1 of 2 roles: It could be transported into ameloblasts (influx) across its proximal membrane to increase energy metabolism or be transported out of ameloblasts (efflux) across its distal membrane into the developing enamel matrix. Based upon findings of citrate in bone and enamel, we hypothesize citrate is secreted and helps to regulate enamel ribbon deposition. The critical roles of ODAPH and SLC13A5 in enamel ribbon formation represent two major gaps in our understanding of amelogenesis. We close these gaps by generating and validating Odaph and Slc13a5 knockout (KO) mice expressing premature stop codons homologous to human AI-causing mutations. We also generate a Slc13a5 knockin (KI) expressing 3 FLAG epitopes on its C-terminus. Two Specific Aims are proposed: SA1: To develop and validate a Slc13a5 KO mouse model homologous to human AI (p.Arg333*) and to generate a Slc13a5FLAG KI mouse for sensitive and specific immunolocalization (IHC). UG3: generate a Slc13a5-/- mouse AI model in C57BL/6J background using CRISPR/Cas9. UG3: generate a Slc13a5FLAG mouse wild-type-tagged model for sensitive and specific localization. UH3: validate the Slc13a5-/- mouse by characterizing its enamel. Validate Slc13a5FLAG mouse by IHC. SA2: To develop and validate an Odaph KO mouse model homologous to human AI (p.Cys43*). UG3: generate an Odaph-/- mouse AI model in C57BL/6J background using CRISPR/Cas9. UH3: validate the Odaph-/- mouse by characterizing its enamel phenotype.