Regulated Tissue Specific BMP Function in Bone Repair

骨形态发生蛋白（BMPs）是一种有效的形态发生素，可促进软骨和成骨分化。这已经在体外、间充质干细胞模型系统内以及当重组BMP被植入或注射到异位、异位或原位位点时的体内得到证实。BMP在骨修复的所有阶段也以升高的水平表达，并且已经显示，当外源性施用时，可以增强愈合反应。假设：BMP在骨折愈合的所有阶段的调节中是至关重要的。为了验证这一假设，本申请中提出的研究将产生采用新实验策略的转基因小鼠 其将转基因的表达限制在软骨或骨中，并允许通过小分子的全身给药对该转基因进行外源性调节。转基因动物将被改造为含有人工转录因子，该因子编码两种蛋白质，一种新的DNA结合结构域和一种激活结构域。这些结构域的表达将由组织特异性启动子（用于软骨的II型胶原蛋白或用于骨的骨钙素）驱动。转录因子将被二聚化剂（雷帕霉素类似物）的外源性施用激活，这使这些结构域接近。当被激活时，它将识别一个独特的启动子，该启动子将驱动BMP-2的过表达或拮抗BMP-2的表达。 BMP通过过度表达Noggin发挥作用。使用这种策略，转基因动物将经历正常的胚胎发育，并且骨折（或者，在项目1的情况下，牵张成骨）将在正常骨骼功能存在的情况下进行。只有在引入二聚化剂后，才能通过这些转基因的过表达诱导功能状态的丧失或获得。然后将在BMP功能改变的条件下，在特定组织和特定时间分析骨折愈合。这些研究将提供有关BMP在关键阶段发挥的具体作用的广泛的新数据 骨折愈合和建立一个强大的模型系统，研究广泛的分子及其对骨骼功能的影响。

Bone morphogenetic proteins (BMPs) are potent morphogens that have been shown to promote chondrogenic and osteogenic differentiation. This has been demonstrated in vitro, within model systems of mesenchymal stem cells, and in vivo when recombinant BMPs have been implanted or injected into ectopic, heterotopic or orthotopic sites. BMPs are also expressed at elevated levels throughout all stages of bone repair and have been shown, when exogenously administered, to enhance the healing response. Hypothesis: BMPs are critical in the regulation of all phases of fracture healing. In order to test this hypothesis, the studies proposed in this application will generate transgenic mice in which a novel experimental strategy is employed that restricts the expression of a transgene to either cartilage or bone and allows for the exogenous regulation of that transgene through systemic administration of a small molecule. Transgenic animals will be engineered to contain an artificial transcription factor encoded in two proteins, a novel DNA binding domain and an activation domain. The expression of these domains will be driven by a tissue-specific promoter (type II collagen for cartilage or osteocalcin for bone). The transcription factor will be activated by the exogenous administration of a dimerizing agent (rapamycin analog), which brings these domains into proximity. When activated, it will recognize a unique promoter which will drive the overexpression of BMP-2 or antagonize BMP function by overexpressing Noggin. Using this strategy, transgenic animals will undergo normal embryological development and fractures (or, in the case of Project 1, distraction osteogensis) will be carried out in the presence of normal skeletal function. Only upon introduction of the dimerizing agent will loss or gain of function states be induced through the overexpression of these transgenes. Fracture healing will then be analyzed in specific tissues and at specific times under conditions in which BMP function is altered. These studies will provide extensive new data concerning the specific roles that BMPs play at critical stages of fracture healing and establish a powerful model system for investigating a wide array of molecules and their effects on skeletal function.