Proect 2 - McMahon

项目 2 - 麦克马洪

• 批准号: 8564863
• 负责人: Marie Demay
• 金额: $ 27.52万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8564863
• 关键词: Age; Aging; Alleles; Automobile Driving; Binding Proteins; Binding Sites; Biology; Bone Diseases; Bone Matrix; Bone Regeneration; Calcium; Cartilage; Cartilage Matrix; Cells; Cephalic; Chondrocytes; Congenital Abnormality; DNA; DNA Binding; Data; Development; Elements; Embryo; Epitopes; Event; Extracellular Matrix; Face; Fracture; Frequencies; Gene Targeting; Genome; Head; Hematopoietic System; Human; Human Genetics; Instruction; Ions; Lateral; Life; Limb structure; Maintenance; Mediating; Mesenchymal; Mesenchymal Stem Cells; Mesoderm; Minerals; Modeling; Molecular; Morphogenesis; Mouse Strains; Mus; Neural Crest; Neural Crest Cell; Online Systems; Organ; Osteoblasts; Osteoclasts; Paraxial Mesoderm; Peptides; Population; Prevalence; Process; Production; Property; Proteins; Regulation; Regulator Genes; Sequence Analysis; Signal Pathway; Signal Transduction; Skeletal Development; Skeleton; Specific qualifier value; Stem cells; Sternum; Therapeutic; Transgenic Mice; Vertebral column; base; bone; cell type; chromatin immunoprecipitation; clavicle; cranium; design; in vivo; insight; knock-down; mouse model; neural plate; progenitor; programs; repaired; repository; skeletal; skeletal disorder; skeletogenesis; transcription factor

PROJECT SUIVIMARY (See instructions): A mineralized endoskeleton supports the mammalian body. Bone also serves as a vital mineral repository and nurtures non-skeletal cell types, notably stem cells of the hematopoietic system. Skeletal anomalies are amongst the most common birth defects, problems in fracture repair are observed in non-union fractures and bone disorders are increasingly prevalent in an ageing population. Two key biosynthetic cell types generate the specific extracellular matrix of the mammalian skeleton: cartilage-secreting chondrocytes and bone secreting osteoblasts. These cells have diverse origins (neural crest, somitic and lateral plate mesoderm), and generate bone by distinct processes: directly from a mesenchymal stem cell (membranous bone) or via a cartilage template (endochondral bone). Mouse and human genetics have identified a number of critical signaling pathways, and three key transcription factors that are master regulators of chondrocyte (Sox9) and osteoblast (Runx2 and Osx) programs. Haploin sufficiency for Sox9 and Runx2 associates with human skeletal deficiencies. While the importance of these factors is well documented, their regulatory actions are not. To this end, we will determine the gene regulatory networks that underpin the regulatory actions of each of these critical skeletal determinants in chondrocyte and osteoblast development. Specific Aim 1: We will use gene-targeting in mouse embryo stem cells to create mice whose endogenous Sox9, Runx2 and Osx proteins are modified by appending a small peptide at their C-terminus. This epitope will enable subsequent analysis of the expression and activity of each factor through common approaches. Specific Aim 2: ChlP-seq analysis will be performed with each tagged protein to directly identify their DNA targets in primary chondrocytes or osteoblasts. Specific Aim 3: Data from microarray-based transcriptional profiling of normal chondrocytes and osteoblasts, or these same populations following knock-down of Sox9, Runx2 and Osx, will be intersected with ChlP-seq data to predict the targets of each regulatory factors actions. Selected regulatory models will be examined in transgenic mouse studies. These data will provide the first genome scale insight into the gene regulatory networks driving mammalian skeletogenesis.

SUIVIMARY 项目（参见说明）： 矿化内骨骼支撑着哺乳动物的身体。骨骼还充当重要的矿物质储存库，并培育非骨骼细胞类型，特别是造血系统的干细胞。骨骼异常是最常见的出生缺陷之一，在不愈合骨折中观察到骨折修复问题，而骨疾病在人口老龄化中越来越普遍。两种关键的生物合成细胞类型产生哺乳动物骨骼的特定细胞外基质：分泌软骨的软骨细胞和分泌骨的成骨细胞。这些细胞具有不同的起源（神经嵴、体节和侧板中胚层），并通过不同的过程产生骨骼：直接来自间充质干细胞（膜骨）或通过软骨模板（软骨内骨）。小鼠和人类遗传学已经确定了许多关键信号通路以及三个关键转录因子，它们是软骨细胞 (Sox9) 和成骨细胞 (Runx2 和 Osx) 程序的主要调节因子。 Sox9 和 Runx2 的单倍体蛋白充足性与人类骨骼缺陷有关。虽然这些因素的重要性已有充分记录，但它们的监管行动却没有。为此，我们将确定支撑软骨细胞和成骨细胞发育中每个关键骨骼决定因素的调控作用的基因调控网络。具体目标 1：我们将在小鼠胚胎干细胞中使用基因打靶技术来创建小鼠，其内源性 Sox9、Runx2 和 Osx 蛋白通过在其 C 末端附加一个小肽进行修饰。该表位将能够通过通用方法对每个因子的表达和活性进行后续分析。具体目标 2：将对每个标记蛋白进行 ChlP-seq 分析，以直接识别原代软骨细胞或成骨细胞中的 DNA 靶标。具体目标 3：来自正常软骨细胞和成骨细胞或敲除 Sox9、Runx2 和 Osx 后的这些相同群体的基于微阵列的转录谱数据，将与 ChlP-seq 数据相交叉，以预测每个调节因子作用的目标。选定的调控模型将在转基因小鼠研究中进行检查。这些数据将为驱动哺乳动物骨骼发生的基因调控网络提供第一个基因组规模的见解。