Gene-targeted mouse models to study the function of versican

描述（由申请人提供）：现在广泛接受细胞外基质（ECM）分子在健康和疾病中都具有调节作用。尽管已被接受，基因工程小鼠仍不能作为研究大硫酸软骨素蛋白聚糖（versican）作用的工具，versican与其结合伙伴透明质酸（hyaluronan）在各种急性和慢性炎症事件中都有关联。我们的实验室参与了动脉粥样硬化和肺部感染的研究，最近还合作研究了哮喘和糖尿病，所有这些疾病都有一个关键的慢性免疫反应，其特征是花青素的表达和积累增加。我们现在建议开发两种基因工程小鼠，这是确定versican在健康和疾病中所起作用的重要工具。在第一个目标中，我们将产生具有versican外显子的小鼠（versicanfl/fl小鼠），这样当与表达可调节的Cre重组酶的小鼠杂交时，可以通过同源重组（versican-/-小鼠）来敲除versican的表达。由于versican基因的破坏会导致胚胎死亡，因此需要有条件的敲除。在第二个目标中，我们将生成一个版本-报告小鼠模型，以确定控制版本表达的机制。我们将产生一个具有驱动荧光蛋白tdTomato表达的versican启动子的小鼠。为了实现这一目标，我们将首先构建一个靶向载体，其中CRE- tdTomato融合蛋白靶向versican基因。这种靶向构建体随后将用于开发版本报告小鼠（versicanTdTR/cspg2）。然后，我们将对versicanTdTR/cspg2小鼠进行繁殖和表征，以证明tdTomato与和versican的表达和积累相关。在第三个目标中，将通过使用versican- tdtomato报告小鼠和versican-/-小鼠来确定versican表达在革兰氏阴性菌肺部炎症反应中的动力学和作用。我们相信，Aim 3中拟议研究的成功完成将提供令人信服的证据，证明蓖麻多糖在肺部感染的炎症反应中起关键作用。如果有可能，这些基因工程小鼠将可用于研究各种各样的病理和损伤，其中versican被认为是一个相关因素，如脊髓损伤，癌变和转移，出生缺陷，心脏瓣膜缺陷和大血管转位，以及伤口修复。在特定炎症事件中明确确定花式多糖的直接作用将刺激未来的治疗策略，将花式多糖作为治疗慢性炎症的手段。PHS 398 （Rev. 11/07）研究计划

DESCRIPTION (provided by applicant): There is now wide acceptance that molecules of the extracellular matrix (ECM) have a regulatory role in both health and disease. Despite this acceptance, genetically engineered mice are still not available for use as tools to study the role of the large chondroitin sulfate proteoglycan, versican, which has been implicated, along with its binding partner hyaluronan, in a wide variety of both acute and chronic inflammatory events. Our laboratories have been involved with the study of atherosclerosis and lung infection, and have more recently collaborated in studies of asthma and diabetes, all of which have a critical chronic immune response that is characterized by the increased expression and accumulation of versican. We now propose to develop two genetically engineered mice, essential tools to determine the role that versican plays in both health and disease. In the first aim, we will generate a mouse with floxed exons of versican (versicanfl/fl mice) such that when crossed with mice expressing a regulatable Cre recombinase, a knock-out of versican expression can be achieved by homologous recombination (versican-/- mice). A conditional knockout is required as disruption of the versican gene results in embryonic lethality. In the second aim we will generate a versican- reporter mouse model to determine the mechanisms controlling the expression of versican. We will generate a mouse with the versican promoter driving the expression of the fluorescent protein, tdTomato. To accomplish this we will first build a targeting vector where a CRE- tdTomato fusion protein is targeted to the versican gene. This targeting construct will then be used to develop the versican-reporter mouse (versicanTdTR/cspg2). We will then breed and characterize the versicanTdTR/cspg2 mice to show that the tdTomato correlates with the expression and accumulation of and versican. In the third aim the kinetics and role of versican expression in the pulmonary inflammatory response to gram-negative bacteria will be determine through the use of versican-tdTomato reporter mice and versican-/- mice. We believe that the successful completion of the proposed studies in Aim 3 will provide compelling evidence that versican plays a key role in the inflammatory response to lung infection. When available, these genetically engineered mice will be available for the study of a wide variety of pathologies and injuries in which versican has been implicated as a relevant factor, such as in spinal cord injury, carcinogenesis and metastasis, birth defects, heart valve defects and great vessel transposition, and wound repair. The unambiguous identification of a direct role for versican in specific inflammatory events will stimulate future therapeutic strategies to target versican as a means of treating chronic inflammation. PHS 398 (Rev. 11/07) Research Plan PUBLIC HEALTH RELEVANCE: This project proposes to develop a conditional knockout of the versican gene in the mouse, as well as a versican reporter mouse for use in future studies to explore the role of versican as a component of the extracellular matrix with which immune cells interact. We believe these models will have wide utility, not just in gaining new insights into the mechanisms of chronic inflammation, but also other fields in which versican is thought to play a role, such as spinal cord injury, cancer and birth defects.