BONE FORMATION IN MICE LACKING THROMBOSPONDIN-2

描述(改编自申请人的摘要)：主要目标 这个修订的SERCA申请的目的是调查Extra的功能 细胞基质蛋白TSP2对小鼠骨形成的调控作用。TSP2 是一种具有多个功能结构域的蛋白质，小鼠具有靶向 TSP2基因的破坏(TSP2-空)具有多个非致命性 异常，包括长骨表型，其特征是 骨内膜骨产量增加。关联中，TSP2-空 骨祖细胞[骨髓基质细胞(MSCs)]数量增加， 显示碱性成纤维细胞生长因子(BFGF)介导的增殖增加。 控制哺乳动物骨形成的局部机制知之甚少； 因此，TSP2空鼠标代表了一个独特的模型，可以在其中研究 细胞外基质蛋白在成骨细胞发育调控中的作用 随后的骨形成。 在具体目标#1中，候选人建议研究体内增殖 TSP2基因缺失小鼠骨髓间充质干细胞的动力学和成骨细胞合成活性。在……里面 特定目标#2 TSP2 I型结构域在碱性成纤维细胞生长因子结合和生长中的作用 将对抑制进行研究。多域结构的意义 在TSP2中的功能尚不清楚。这一目标将测试类型I TSP2的重复是bFGF介导的负性调节所必需的 扩散。TSP2抑制碱性成纤维细胞生长因子介导的MSC的机制 将在具体目标3中检查增殖，特别是TSP2 结合碱性成纤维细胞生长因子可抑制信号转导。在本提案的第二阶段(具体 目标#4)，申请者将生成并评估新的 小鼠模型。缺乏I型重复序列的小鼠将通过替换 带有Thbs2基因的内源性Thbs2等位基因缺乏编码区 我重复的类型。通过对比研究“敲门”的表型 对于TSP2缺失的小鼠，I型在体内重复的重要性 TSP2的功能将被确定。 威斯康星大学是美国首屈一指的生物医学研究机构之一， 它的教职员工包括一大批ECM调查人员，一位备受尊敬的 整形外科研究小组，以及一个非常活跃的转基因计划。 Ladiges(第二导师)。主要导师博恩斯坦博士一直是 自20世纪70年代初以来杰出的ECM调查员之一和他的 该领域的杰出贡献者已被美国国立卫生研究院表彰 获奖。该实验室目前正在参与产生和分析 一批ECM基因敲除和转基因小鼠。 申请者在普渡大学获得硕士学位，经过两年的学习 马术实践，是他在威斯康星大学博士项目的第三年。通过 他完成了对TSP2在骨内膜骨形成中的作用的分析 将掌握分子和细胞生物学、基因打靶和 小鼠病理生物学，主要研究细胞外基质生物学和骨科 科学。应聘者的职业目标是获得一份学术职位 比较形态学讲师，并开发一项研究计划， 建立新的小鼠模型来研究ECM蛋白在脑内的功能 整形外科的愈合和重建。

DESCRIPTION (Adapted from the applicant's abstract): The principal objective of this revised SERCA application is to investigate the function of the extra cellular matrix (ECM) protein TSP2 in regulating murine bone formation. TSP2 is a protein with multiple functional domains and mice with a targeted disruption of the TSP2 gene (TSP2-null) have a number of non-lethal abnormalities, including a long-bone phenotype that is characterized by increased endosteal bone production. In association, TSP2-null osteoprogenitors [marrow stromal cells (MSCs)] are increased in number and show increased basic fibroblast growth factor (bFGF)-mediated proliferation. Local mechanisms that control mammalian bone formation are poorly understood; thus the TSP2 null mouse represents a unique model in which to study the function of an ECM protein in the regulation of osteoblast development and subsequent bone formation. In Specific Aim #1, the candidate proposes studying the in vivo proliferation dynamics and osteoblast synthetic activity of MSCs from TSP2-null mice. In Specific Aim #2 the role of the TSP2 type I domain in bFGF binding and growth inhibition will be studied. The significance of the multiple domain structure in the function of TSP2 is unknown. This aim will test whether the type I repeats of TSP2 are necessary to impart negative regulation of bFGF-mediated proliferation. The mechanism by which TSP2 inhibits bFGF-mediated MSC proliferation will be examined in Specific Aim #3, specifically, whether TSP2 binding to bFGF inhibits signaling. In phase two of this proposal (Specific Aim #4), the applicant will generate and evaluate the phenotype of a new murine model. Mice lacking the type I repeats will be generated by replacing the endogenous Thbs2 allele with a Thbs2 gene lacking the coding region for the type I repeats. By studying the phenotype of the "knockin," in comparison to the TSP2-null mice, the importance of the type I repeats in the in vivo function of TSP2 will be determined. UW is one of the premier biomedical research institutions in the country and its faculty includes a large group of ECM investigators, a highly-regarded orthopedic research group, and a very active transgenic program headed by Dr. Ladiges (the secondary mentor). The primary mentor, Dr. Bornstein, has been one of the pre-eminent ECM investigators since the early 1970s and his outstanding contributors to the field have been recognized with an NIH MERIT award. The laboratory is currently involved in the generation and analysis of a number of ECM knockout and transgenic mice. The applicant earned a MS from Purdue University, following two years of equine practice, and is in his third year of a PhD program at UW. By completing an analysis of the role of TSP2 in endosteal bone formation, he will master techniques in molecular and cell biology, gene targeting, and mouse pathobiology, directed at the study of ECM biology and orthopedic science. The applicant's career goal is to obtain an academic position as an instructor of comparative morphology and to develop a research program that generates new murine models to study the function of ECM proteins in orthopedic healing and remodeling.