CELL AND GENETIC APPROACHES TO ENAMEL BIOMIMETICS

牙釉质仿生学的细胞和遗传学方法

基本信息

批准号：
7223470
负责人：
Malcolm L. Snead
金额：
$ 37.05万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7223470
关键词：
Ameloblasts Animal Model Animals Appendix Architecture Atomic Force Microscopy Behavior Biological Biomechanics Biomimetics Cell Communication Cell Cycle Cells Code Communicable Diseases Condition Coupled Cultured Cells DSPP gene Data Dental Dental Enamel Dental caries Dentin Deposition Devices Ectoderm Enamel Formation Enamel Organ Engineering Environment Epithelium Extracellular Matrix Extracellular Matrix Proteins Failure Fracture Gene Expression Gene Targeting Generations Genes Genetic Germ Lines Habits Hardness Human In Vitro Interphase Knock-in Mouse Knock-out Knowledge Link Maps Measures Mechanics Minerals Molecular Biology Techniques Molecular Profiling Mus Normal tissue morphology Organ Organ Model Outcome Personal Satisfaction Pharmaceutical Preparations Phase Phenotype Play Positioning Attribute Process Production Property Protein Engineering Proteins Public Health Relative (related person)Research Research Personnel Rodent Role Series Structure Structure-Activity Relationship Tertiary Protein Structure Testing Time Tissue Engineering Tissues Tooth structure Transgenes Transgenic Animals Transgenic Mice Transgenic Organisms Upper arm Work amelogenin base biomineralization design enamel matrix proteins extracellular functional genomics gain of function genetic manipulation homologous recombination in vivo insight knowledge base light microscopy light scattering loss of function mouse genome nanoscale novel physical property protein expression protein function reconstitution research study retinal rods self assembly stem tool

项目摘要

DESCRIPTION (provided by applicant): Deciphering structure-function relationships is the fundamental underpinning of our biomedical knowledge and provides the basis for our ability to create novel materials and drugs by knowledge-based design. Powerful tools aimed towards deciphering structure-function outcomes is germ-line genetic manipulation in mice. Enamel is a composite tissue with unique material properties that are owed to its biological fabrication. Ameloblast cells create an enamel protein matrix that serves to control crystallite habit and the organization of crystallite bundles. We hypothesize that the structure of enamel, from the nanoscale to the macroscale, is the outcome of the function(s) of critical domains within the ameloblastin protein. Ablating protein expression by aknock out shows the requirement for amelogenin and ameloblastin protein for normal enamel formation, although the loss of function does not provide insight into the underlying mechanism of protein action. The loss of ameloblastin disrupts ameloblast cell interactions with the matrix resulting in ameloblasts detaching from the forming matrix and re-entering the cell cycle. Here the process of enamel formation is so severely disrupted that the underlying function(s) of the ameloblastin protein in normal tissue formation is difficult to discern. We hypothesize that using a knock-in strategy will allow us to link the identity of a specific ameloblastin protein domain(s) to the function(s) it contributes to formation of the enamel tissue. We propose the use of homologous recombination coupled with protein engineering of a human ameloblastin minigene in order to modify the mouse genome and to decipher the function(s) that human ameloblastin domain(s) contribute to enamel biomineralization. Insights from similar work performed for the amelogenin protein suggests that the approach of knock-in gene targeting of a minigene preserves the qualitative and quantitative aspects of gene expression while permitting the use of an ameloblastin minigene designed to express an engineered ameloblastin protein that will allow insights into the function of the engineered protein. Functional changes will be measured by alteration to stereotypic enamel architecture and by analysis of the material properties of the enamel in the knock in condition compared to wild type animals. This experimental strategy will contribute significant information to functional genomics and to further understanding of the only ectoderm-derived biomineralized tissue in the vertebrate body. Preliminary data from this group on the use of a similar strategy using a knock-in engineered amelogeninminigene suggest that this approach will yield novel insights into the structure-function relationship for the ameloblastin protein, the second most abundant protein contributing to enamel organic matrix assembly and biomineralization. Humanizing rodent enamel will also yield a new animal model that would be useful to investigators exploring the most prevalent infectious disease of mankind, dental caries. PUBLIC HEALTH RELEVANCE: The function(s) for the second most abundant protein of the forming mammalian enamel matrix is not known. Knocking out ameloblastin demonstrated that it plays an essential role, as enamel did not form in the absence of ameloblastin. Here, we map the function(s) of ameloblastin domains to the production of an enamel matrix required to control enamel biomineralization thus humanizing an animal model used to study caries, the most prevalent infectious disease of humankind.

描述（由申请人提供）：解密结构 - 功能关系关系是我们生物医学知识的基本基础，并为我们通过基于知识的设计创建新型材料和药物的能力为基础提供了基础。旨在破译结构功能结果的强大工具是小鼠种系遗传操作。搪瓷是一种复合组织，其独特的材料特性归功于其生物制造。成熟细胞产生牙釉质蛋白基质，该基质可控制结晶石习惯和结晶石束的组织。我们假设搪瓷的结构从纳米尺度到宏观，是杏仁核细胞蛋白中关键域功能的结果。通过AKNOCK OUT的消融蛋白表达表明对正常牙釉质形成的蛋白质蛋白和蛋白质蛋白的需求，尽管功能的丧失并不能洞悉蛋白质作用的潜在机制。氨基蛋白细胞蛋白的丧失破坏了与基质的成成布细胞相互作用，从而导致成成木与形成矩阵脱离并重新进入细胞周期。在这里，牙釉质形成的过程被严重破坏，以至于很难辨别出正常组织形成中杏仁蛋白蛋白的潜在功能。我们假设使用敲门策略将使我们能够将特定的蛋白蛋白蛋白结构域的身份联系起来与牙釉质组织的形成有助于该功能。我们提出了同源重组的使用，再加上人氨基细胞蛋白小蛋白的蛋白质工程，以修改小鼠基因组并破译该功能，即人amelomeloblastin结构域有助于搪瓷生物矿化。针对阿米他蛋白蛋白所做的类似工作的见解表明，敲入基因靶向微岛的方法可以保留基因表达的定性和定量方面，同时允许使用旨在表达工程化的无纤维母细胞蛋白的无蛋白质细胞蛋白微生蛋白，从而允许启用工程蛋白质的功能。功能变化将通过改变刻板印象牙釉质体系结构和分析牙釉质的材料特性来衡量功能变化与野生型动物相比，状态下的状态。该实验策略将为功能基因组学提供重要的信息，并进一步了解脊椎动物中唯一的外胚层生物矿化组织。该小组使用类似策略的初步数据使用敲门工程的amelogeninminigene表明，这种方法将产生对杏仁糖蛋白蛋白的结构功能关系的新见解，阿素细胞蛋白是第二大最丰富的蛋白质，这是搪瓷有机基质组装和生物矿化的第二大蛋白质。人性化的啮齿动物搪瓷还将产生一种新的动物模型，该模型对探索最普遍的人类龋齿传染病的研究人员有用。公共卫生相关性：尚不清楚形成哺乳动物搪瓷基质的第二大蛋白质的功能。淘汰氨基蛋白细胞蛋白表明它起着至关重要的作用，因为搪瓷没有在没有蛋白蛋白的情况下形成。在这里，我们将蛋白细胞蛋白结构域的函数映射到控制搪瓷生物矿化所需的牙釉质基质的产生人性化用于研究龋齿的动物模型，这是人类最普遍的传染病。