CELL AND GENETIC APPROACHES TO ENAMEL BIOMIMETICS

描述（由申请人提供）：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了基础。旨在破译结构-功能结果的强大工具是在小鼠中进行种系遗传操作。牙釉质是一种复合组织，具有独特的材料特性，这要归功于它的生物制造。成釉细胞产生釉质蛋白基质，用于控制晶体习性和晶体束的组织。我们假设，牙釉质的结构，从纳米尺度到宏观尺度，是成釉蛋白关键结构域功能的结果。通过敲除的消融蛋白表达表明，正常的牙釉质形成需要成釉素和成釉素蛋白，尽管功能的丧失并不能提供蛋白质作用的潜在机制。成釉素的缺失破坏了成釉细胞与基质的相互作用，导致成釉细胞脱离形成基质，重新进入细胞周期。在这里，牙釉质形成的过程被严重破坏，以至于成釉蛋白在正常组织形成中的潜在功能难以辨别。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的身份与其对牙釉质组织形成的作用联系起来。我们提出利用同源重组结合蛋白工程的方法来修饰小鼠基因组，并破译人类成釉酶结构域在牙釉质生物矿化中的作用。对成釉素蛋白进行的类似研究表明，敲入基因靶向小基因的方法保留了基因表达的定性和定量方面，同时允许使用设计成釉素小基因来表达工程成釉素蛋白，从而可以深入了解工程蛋白的功能。通过对牙釉质结构的改变和对牙釉质材料特性的分析，可以测量牙釉质的功能变化

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.