CELL AND GENETIC APPROACHES TO ENAMEL BIOMIMETICS

描述(申请人提供)：破译结构-功能关系是我们生物医学知识的基础，并为我们通过基于知识的设计创造新材料和药物的能力提供基础。旨在破译结构-功能结果的强大工具是小鼠的生殖系遗传操作。牙釉质是一种具有独特材料特性的复合组织，其生物构造具有独特的性质。成釉细胞产生一种釉质蛋白基质，用于控制微晶习性和微晶束的组织。我们假设，釉质的结构，从纳米尺度到宏观尺度，是成釉蛋白中关键结构域功能(S)的结果。Anock Out的消融蛋白表达表明，正常的釉质形成需要成釉蛋白和成釉蛋白，尽管功能的丧失并不能深入了解蛋白质作用的潜在机制。成釉蛋白的丢失扰乱了成釉细胞与基质的相互作用，导致成釉细胞脱离形成的基质，重新进入细胞周期。在这里，釉质形成过程被严重破坏，以至于成釉蛋白在正常组织形成中的潜在功能(S)很难辨别。我们假设，使用敲入策略将使我们能够将特定成釉蛋白结构域(S)的身份与其促进釉质组织形成的功能(S)联系起来。我们提出了利用同源重组和蛋白质工程相结合的方法对小鼠基因组进行修饰，并破译了人成釉蛋白结构域(S)在釉质生物矿化中的作用(S)。对釉原蛋白进行的类似工作的见解表明，敲入基因打靶的方法保留了基因表达的定性和定量方面，同时允许使用旨在表达工程成釉蛋白的造釉蛋白微基因，这将使人们能够深入了解工程蛋白的功能。功能变化将通过改变刻板的牙釉质结构和分析牙釉质的材料特性来衡量 与野生动物相比，它们处于敲打状态。这一实验策略将为功能基因组学和进一步了解脊椎动物体内唯一的外胚层来源的生物矿化组织提供重要信息。来自该小组的使用敲入工程造釉蛋白迷你基因的类似策略的初步数据表明，这种方法将对成釉蛋白的结构与功能关系产生新的见解，成釉蛋白是第二丰富的蛋白质，有助于釉质有机基质组装和生物矿化。将啮齿动物的牙釉质人性化还将产生一种新的动物模型，这将有助于研究人员探索人类最普遍的传染病--龋齿。 公共卫生相关性：哺乳动物牙釉质基质形成中第二丰富的蛋白质(S)的功能尚不清楚。敲除成釉蛋白表明它起着至关重要的作用，因为在没有成釉蛋白的情况下，釉质不会形成。在这里，我们将成釉蛋白结构域的功能(S)映射到控制釉质生物矿化所需的釉质基质的产生上 使用于研究龋病的动物模型人性化，龋病是人类最常见的传染病。

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.