Development and Validation of Novel Amelogenesis Models

新型釉质生成模型的开发和验证

• 批准号: 9796443
• 负责人: JAN Ching Chun HU
• 金额: $ 31.2万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9796443
• 关键词: Acetyl Coenzyme A; Acids; Ameloblasts; Amelogenesis; Amelogenesis Imperfecta; Antibodies; Bicarbonates; Biomimetics; Brain; CRISPR/Cas technology; Caring; Citrate (si)-Synthase; Citrates; Citric Acid Cycle; Defect; Dental; Dental Enamel; Dental Enamel Hypoplasia; Dentin; Deposition; Development; Dimensions; Disease; Disease Progression; Dissection; Distal; Enamel Formation; Energy Metabolism; Ensure; Epitopes; Exhibits; Extracellular Space; Gene Expression; Gene Proteins; Genes; Graph; Hardness; Heterozygote; Human; Immunohistochemistry; In Situ Hybridization; In Vitro; Incisor; Individual; Inherited; Ions; Knock-in; Knock-in Mouse; Knock-out; Knockout Mice; Left; Liver; Membrane; Microscopy; Minerals; Missense Mutation; Mitochondrial Matrix; Modeling; Mus; Mutation; Nonsense Codon; Oral; Organ; Oxaloacetates; Patients; Pattern; Persons; Phase; Phenotype; Play; Positioning Attribute; Predisposition; Process; Proteins; Radio; Reaction; Research; Research Proposals; Role; Surface; Syndrome; Terminator Codon; Thick; Tooth Tissue; Tooth structure; Translations; Validation; Water; Wild Type Mouse; ameloblastin; base; biomineralization; bone; citrate carrier; design; epileptic encephalopathies; extracellular; human disease; improved; infancy; insight; loss of function; malformation; mouse model; novel; outcome forecast; premature; retinal rods

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.

抽象的 该研究计划重点关注牙釉质矿物带起始的釉质形成的分泌阶段 在牙本质矿物质上并伸长，直到牙釉质层达到其最终尺寸。许多基因会导致遗传 牙釉质畸形（釉质发育不全，AI）。分泌阶段必需的基因缺陷 通常会导致牙釉质变薄（发育不良）且表面粗糙。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 个导致牙釉质发育不全的人类 AI 基因， 但重点关注那些与细胞外牙釉质基质最直接相关的。五个基因编码分泌 基质蛋白：ENAM、AMBN、AMELX、MMP20 和 ODAPH。除了 ODAPH（以前称为 C4orf26）之外，这些 基因/蛋白质具有良好的特征。有 2 个关键的分泌阶段离子转运蛋白：SLC4A4 (NCBe1； 将碳酸氢盐输送到牙釉质基质中）和 SLC13A5。已知碳酸氢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 牙釉质畸形尚不清楚。 软组织（肝、脑）和硬组织（骨、牙齿）都需要 SLC13A5。令人惊讶的是~80%的柠檬酸盐 身体在骨头里。柠檬酸盐是柠檬酸循环的一部分，其中柠檬酸盐由草酰乙酸、乙酰基- 辅酶 A 和水在线粒体基质内的柠檬酸合酶催化下发生反应。可能是柠檬酸盐 发挥 2 个角色之一：它可以穿过其近端膜转运到成釉细胞（流入）中，以增加 能量代谢或从成釉细胞（流出）穿过其远端膜输送到发育中的 牙釉质基质。根据骨骼和牙釉质中柠檬酸盐的发现，我们假设柠檬酸盐被分泌并有助于 调节釉质带沉积。 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理解的两个主要差距。我们通过生成和缩小这些差距 验证 Odaph 和 Slc13a5 敲除 (KO) 小鼠表达与人类同源的过早终止密码子 人工智能引起的突变。我们还生成了 Slc13a5 敲入 (KI)，在其 C 末端表达 3 个 FLAG 表位。 提出了两个具体目标： SA1：开发并验证与人类 AI (p.Arg333*) 同源的 Slc13a5 KO 小鼠模型，并 生成 Slc13a5FLAG KI 小鼠，用于敏感和特异性免疫定位 (IHC)。 UG3：使用 CRISPR/Cas9 在 C57BL/6J 背景下生成 Slc13a5-/- 小鼠 AI 模型。 UG3：生成 Slc13a5FLAG 小鼠野生型标记模型，用于敏感和特异性定位。 UH3：通过表征其牙釉质来验证 Slc13a5-/- 小鼠。通过 IHC 验证 Slc13a5FLAG 小鼠。 SA2：开发并验证与人类 AI (p.Cys43*) 同源的 Odaph KO 小鼠模型。 UG3：使用 CRISPR/Cas9 在 C57BL/6J 背景下生成 Odaph-/- 小鼠 AI 模型。 UH3：通过表征其牙釉质表型来验证 Odaph-/- 小鼠。