Analysing the Role of Growth Plate Specific Genes in Endochondral Ossification by Specific Overexpression or Deletion under the BAC Col10a1 Promoter

软骨内骨化引起的软骨-骨转化是一个高度复杂的过程，不仅决定着长骨、肋骨和椎骨的纵向生长，而且在骨折骨痂愈合、骨赘形成和软骨组织工程中起着至关重要的作用。在这个过程中的决定性步骤是生长板中的软骨细胞成熟和肥大，然后诱导肥大的软骨矿化、凋亡和吸收，随后是骨髓侵袭。这些事件受多种信号通路和转录因子协同作用的调控，这些信号通路和转录因子受多种生长因子和激素的控制。为了阐明软骨内成骨相关基因的特定功能，我们设计了一种胶原X特异的BAC(细菌人工染色体)重组工程系统，该系统允许报告基因和其他与肥大软骨相关的基因在转基因小鼠的Co110a1启动子下特异性和强劲地过表达。这个项目的具体目标是培育一只Co110a1-BAC-Cre小鼠，它将允许突变小鼠生长板中特定基因的缺失，方法是移除位于1oxP位点两侧的外显子，主要是(L积分、PTHrP和(-连环蛋白)。其次，转录因子Sox9和激素样因子PTHrP将在BAC-Co110a1转基因小鼠中过表达，以阐明它们在软骨内成骨中的特殊功能。我们认为，这种Co110a1特异性的BAC重组工程系统将为阐明和操纵软骨内成骨的复杂过程提供一个强大而特异的新工具。

Cartilage-bone transition by endochondral ossification is a highly complex process which determines not only longitudinal growth of long bones, ribs and vertebrae, but also plays a critical role in bone fracture callus healing, osteophyte formation and cartilage tissue engineering. Decisive steps in this process are maturation and hypertrophy of chondrocytes in the growth plate before inducing mineralisation, apoptosis and resorption of hypertrophic cartilage followed by bone marrow invasion. These events are regulated by synergistic interactions of several signalling pathways and transcriptions factors controlled by a multitude of growth factors and hormones. In order to elucidate specific functions of genes presumably involved in endochondral ossification, we designed a collagen X specific BAC (Bacterial Artificial Chromosome) recombineering system which allows specific and robust overexpression of reporter genes and other genes of interest in hypertrophic cartilage under the authentic Co110a1 promoter in transgenic mice. The specific aim of this project is to generate a Co110a1-BAC-CRE mouse which will allow specific gene deletion in the growth plate of mutated mice by removing exons flanked by 1oxP sites, primarily (l integral, PTHrP and (-catenin. Second, the transcription factor Sox9 and the hormone-like factor PTHrP, both know to delay chondrocyte maturation, will be overexpressed in BAC –Co110a1 transgenic mice in order to elucidate their specific function in endochondral ossification. We propose that this Co110a1-specific BAC recombineering system will provide a powerful and specific new tool to elucidate and to manipulate the complex process of endochondral ossification.