Strategy to assess the function of osteocyte restricted genes

描述（由申请人提供）： 骨细胞是从成骨细胞分化而来的终末分化细胞，成骨细胞被包埋在其自身的基质中。骨细胞通过建立树突状突起的相互连接的小管网络而存活，通过该网络它们与其他骨细胞和成骨细胞谱系细胞进行通信。众所周知，骨组织具有使用骨细胞作为机械感觉细胞响应于机械应变而改变其质量和结构的能力。 鉴定这些过程中涉及的基因需要分离纯的骨细胞群体，并与成骨细胞谱系的其他成熟阶段的细胞的基因表达谱进行比较。在这个项目中，我们建议评估一个孤立的骨细胞群体的基因表达谱，并将其基因表达与成骨细胞转化为骨细胞阶段的基因表达进行对比。 将使用膜内骨化过程中小鼠颅骨成骨细胞分化的定义模型。将使用来自携带由牙本质基质蛋白1（GFP-1）启动子驱动的GFP托帕石的小鼠的顶骨的连续酶消化分离所有成熟阶段的细胞。 该启动子已被证明驱动GFP表达到被截留在基质（前骨细胞）和骨细胞中的细胞。为了从成骨细胞中分离骨细胞，将DMP 1-tpz转基因小鼠（骨细胞特异性标记）与pOBCol 2. 3GFP-saphirre小鼠（在成骨细胞和骨细胞中有活性的转基因）杂交。 将从颅骨酶促释放的细胞进行荧光分选，以基于双色系统获得成骨细胞和骨细胞的分离群体。 除了基因表达分析，这项研究将解决在骨细胞阶段高度表达的基因的作用，并有可能在骨细胞生物学中发挥重要作用。分析其基因表达谱将为更好地理解调节骨量的机制提供重要信息。我们将评估逆转录病毒过表达或通过siRNA下调感兴趣基因的影响。这项研究的结果将为一个更大的提议奠定基础，该提议将包括测试通过这项分析鉴定的特定基因的缺乏或过表达的体内影响。

DESCRIPTION (provided by applicant): Osteocytes are terminally differentiated cells derived from osteoblasts that become entrapped in their own matrix. Osteocytes survive by establishing a mutually connected canalicular network of dendritic processes by which they communicate with other osteocytes and other osteoblast lineage cells. It is well known that bone tissue has the capacity to alter its mass and structure in response to mechanical strain using osteocytes as mechanosensory cells. Identification of genes involved in these processes requires isolation of a pure population of osteocytes and a comparison to the gene expression profile to cells at other maturation stages of the osteoblast lineage. In this project we propose to evaluate the gene expression profile of an isolated osteocyte population and contrast its gene expression to osteoblasts as they transition into the osteocyte stage. A defined model of mouse calvarial osteoblast differentiation during the process of intramembranous ossification will be utilized. Cells of all maturation stages will be isolated using a sequential enzymatic digestion of parietal bones derived from mice harboring GFPtopaz driven by the dentin matrix protein 1 (DMP-1) promoter. This promoter has been shown to drive the expression of GFP to cells that are becoming entrapped in the matrix (preosteocytes) and in the osteocytes. To separate osteocytes from osteoblasts, DMP1-tpz transgenic mice (osteocyte specific marker) will be crossed with pOBCol2.3GFP-saphirre mice (transgene active in osteoblasts and osteocytes). Enzymatically released cells from calvaria will be fluorescence-sorted to obtain isolated populations of osteoblasts and osteocytes on the basis of the two-color system. In addition to gene expression analysis, this study will address the role of genes that are highly expressed at the osteocyte stage and have the potential to play an important role in osteocyte biology. The analysis of their gene expression profile will provide important information for a better understanding of the mechanisms that regulate bone mass. We will evaluate the effects of retroviral overexpression or downregulation of genes of interest by siRNA. The results of this study will generate the basis for a larger proposal that will include testing the in vivo effects of deficiency or overexpression of particular genes identified by this analysis.