Gene-targeted mouse models to study the function of versican

描述(申请人提供)：现在人们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。尽管这一点得到了认可，但基因工程小鼠仍然不能用作研究大的硫酸软骨素蛋白多糖(Verscan)的工具，该蛋白多糖与其结合伙伴透明质酸在各种急性和慢性炎症事件中都有牵连。我们的实验室一直参与动脉粥样硬化和肺部感染的研究，最近还在哮喘和糖尿病的研究中进行了合作，所有这些都具有关键的慢性免疫反应，其特征是Versican的表达和积累增加。我们现在提议开发两种基因工程小鼠，这是确定Verscan在健康和疾病中扮演的角色的基本工具。在第一个目标中，我们将产生一种带有versica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(verscan-/-小鼠)实现verscan表达的敲除。需要进行有条件的基因敲除，因为verscan基因的破坏会导致胚胎死亡。在第二个目的中，我们将建立Verscan-Report小鼠模型，以确定Verscan表达的控制机制。我们将产生一只带有多功能启动子的小鼠，该启动子驱动荧光蛋白tdTomato的表达。为了实现这一点，我们将首先构建一个靶向载体，其中Cre-tdTomato融合蛋白靶向verscan基因。然后，这种靶向构建物将被用于开发通用报告鼠(versicanTdtr/cspg2)。然后，我们将培育和鉴定versicanTdtr/cspg2小鼠，以表明tdTomato与和versican的表达和积累相关。在第三个目标中，将通过Verscan-tdTomato报告小鼠和Verscan-/-小鼠来确定Verscan在革兰氏阴性菌肺炎症反应中的表达动力学和作用。我们相信，目标3中拟议研究的成功完成将提供令人信服的证据，证明Verscan在肺部感染的炎症反应中发挥关键作用。一旦可用，这些基因工程小鼠将可用于研究多种病理和损伤，如脊髓损伤、癌症发生和转移、出生缺陷、心脏瓣膜缺陷和大血管移位以及伤口修复。明确地确认万西康在特定炎症事件中的直接作用将刺激未来的治疗策略，将万西康作为治疗慢性炎症的一种手段。小灵通398(11/07版)研究计划 与公共卫生相关：该项目建议在小鼠中开发一种有条件的万西卡基因敲除，以及一种万西卡报告小鼠，用于未来的研究，以探索万西卡作为免疫细胞相互作用的细胞外基质的组成部分所起的作用。我们相信，这些模型将具有广泛的实用价值，不仅在获得对慢性炎症机制的新见解方面，而且在多西肯被认为在其中发挥作用的其他领域，如脊髓损伤、癌症和出生缺陷。

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.