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.

摘要 这项研究计划的重点是分泌阶段的釉质矿物质带开始 在牙本质矿物质上延伸，直到釉质层达到其最终尺寸。许多基因导致遗传 釉质畸形（釉质形成，AI）。分泌期所必需的基因缺陷 典型地导致具有粗糙表面的较薄（发育不良）釉质。牙釉质形成在一个明确的细胞外 由成釉细胞建立和改变的空间。我们引用了19个人类AI基因导致釉质发育不全， 而是集中在与细胞外釉质基质最直接相关的那些。五个基因编码分泌型 基质蛋白：ENAM、AMBN、AMELX、MMP 20和ODAPH。除了ODAPH（以前的C4 orf 26）， 基因/蛋白质被很好地表征。分泌期的离子转运蛋白有两个：SLC 4A 4（NCBe 1; 将碳酸氢盐转运到釉质基质中）和SLC 13 A5。众所周知，重碳酸盐可以中和产生的酸 但为什么SLC 13 A5编码的柠檬酸盐转运蛋白（NaCT）的缺失会导致严重的 牙釉质畸形是未知的。 SLC 13 A5在软组织（肝脏，大脑）和硬组织（骨骼，牙齿）中是必需的。令人惊讶的是， 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- 辅酶A和水在由线粒体基质内的柠檬酸合酶催化的反应中。可能是柠檬酸盐 发挥2种作用之一：它可以通过成釉细胞近端膜转运到成釉细胞（内流）， 能量代谢或被运输出成釉细胞（流出）穿过其远端膜进入发育中的 釉质基质基于骨和釉质中柠檬酸盐的发现，我们假设柠檬酸盐是分泌的， 以调节釉带沉积。ODAPH和SLC 13 A5在釉带形成中的关键作用 代表了我们对釉质形成理解的两个主要空白。我们通过生成和 验证表达与人类同源的提前终止密码子的Odaph和Slc 13 a5敲除（KO）小鼠 导致人工智能的突变我们还产生了在其C-末端表达3个FLAG表位的Slc 13 a5敲入（KI）。 提出了两个具体目标： SA 1：开发并验证与人AI（p.Arg333*）同源的Slc 13 a5 KO小鼠模型， 产生用于敏感和特异性免疫定位（IHC）的Slc 13 a5 FLAG KI小鼠。 UG 3：使用CRISPR/Cas9在C57 BL/6 J背景中生成Slc 13 a5-/-小鼠AI模型。 UG 3：生成Slc 13 a5 FLAG小鼠野生型标记模型，用于敏感和特异性定位。 UH 3：通过表征其牙釉质来验证Slc 13 a5-/-小鼠。免疫组化法检测Slc 13 a5 FLAG小鼠。 SA 2：开发并验证与人AI（p.Cys43*）同源的Odaph KO小鼠模型。 UG 3：使用CRISPR/Cas9在C57 BL/6 J背景中生成Odaph-/-小鼠AI模型。 UH 3：通过表征其釉质表型来验证Odaph-/-小鼠。