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 这项研究建议着重于搪瓷矿物质丝带的分泌阶段 在牙本质矿物质上并拉长，直到搪瓷层达到其最终尺寸。许多基因导致继承 搪瓷畸形（Amelogeneseres Interfecta，AI）。分泌阶段所需的基因缺陷 通常会导致具有粗糙表面的较薄（低塑性）搪瓷。牙釉质形式在定义的细胞外 由成成布建立和修改的​​空间。我们引用了19个引起牙釉质发育不全的人AI基因， 但是专注于与细胞外搪瓷基质最直接相关的人。五个基因编码分泌 基质蛋白：ENAM，AMBN，AMELX，MMP20和ODAPH。除了ODAPH（以前是C4ORF26），这些 基因/蛋白质是良好的。有2个关键分泌阶段离子转运蛋白：SLC4A4（NCBE1； 将碳酸氢盐运输到搪瓷基质中）和SLC13A5。已知碳酸氢盐可以中和产生的酸 通过矿物沉积，但是为什么缺乏由Slc13a5编码的柠檬酸盐转运蛋白转运蛋白（NACT）会导致严重 搪瓷畸形未知。 SLC13A5在软（肝脏，脑）和硬（骨，牙齿）组织中需要。出人意料的是，所有柠檬酸盐中的80％ 身体在骨头。柠檬酸盐是柠檬酸循环的一部分，其中柠檬酸盐是由草乙酸，乙酰基产生的 辅酶A和在线粒体基质中由柠檬酸盐合酶催化的反应中的水。柠檬酸盐可能 角色中有1个角色：它可以将其转运到近端膜上的成成布（涌入）中以增加 能量代谢或从其远端膜中输出的成年细胞（外排）到发育中 搪瓷矩阵。根据柠檬酸盐在骨和搪瓷中的发现，我们假设柠檬酸盐是分泌的，有助于 调节搪瓷色带沉积。 ODAPH和SLC13A5在搪瓷色带形成中的关键作用 在我们理解不稳态时，代表了两个主要差距。我们通过生成和 验证ODAPH和SLC13A5敲除（KO）小鼠表达与人同源的早产停止密码子 引起AI的突变。我们还产生了SLC13A5敲击蛋白（Ki），其C末端表达了3个标志表位。 提出了两个具体目标： SA1：开发和验证与人AI同源（P.Arg333*）的SLC13A5 KO小鼠模型和 生成SLC13A5FLAG KI小鼠进行敏感和特定的免疫定位（IHC）。 UG3：使用CRISPR/CAS9生成C57BL/6J背景中的SLC13A5 - / - 鼠标AI模型。 UG3：生成用于敏感和特定定位的SLC13A5Flag小鼠野生型模型。 UH3：通过表征其搪瓷来验证SLC13A5 - / - 鼠标。通过IHC验证SLC13A5FLAG鼠标。 SA2：开发和验证与人AI同源的ODAPH KO小鼠模型（P.Cys43*）。 UG3：使用CRISPR/CAS9在C57BL/6J背景中生成ODAPH - / - 鼠标AI模型。 UH3：通过表征其搪瓷表型来验证ODAPH - / - 小鼠。