Gene-targeted mouse models to study the function of versican

研究多功能蛋白聚糖功能的基因靶向小鼠模型

• 批准号: 7872666
• 负责人: Charles Wayne Frevert
• 金额: $ 17.85万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-11 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7872666
• 关键词: 4-Hydroxy-Tamoxifen; Acute; Affect; Alleles; Animals; Asthma; Atherosclerosis; Automobile Driving; Binding; Biology; Breeding; C-terminal; Cardiovascular Diseases; Cell Adhesion; Cell Adhesion Molecules; Cells; Chimeric Proteins; Chondroitin Sulfate Proteoglycan; Chronic; Chronic lung disease; Congenital Abnormality; Defect; Development; Diabetes Mellitus; Disease; Effectiveness; Embryo; Event; Exons; Extracellular Matrix; Future; Gene Targeting; Generations; Genes; Genetically Engineered Mouse; Glycosaminoglycans; Goals; Gram-Negative Bacteria; Growth Factor; Health; Heart Valves; Hyaluronan; Immune; Immune response; Infection; Inflammation; Inflammatory; Inflammatory Response; Injury; Kinetics; Knock-out; Knockout Mice; Laboratories; Lung; Lung diseases; Malignant Neoplasms; Modeling; Mus; Names; Neoplasm Metastasis; Neutrophil Infiltration; Pathogenesis; Pathology; Play; Process; Proteins; Proteoglycan; RNA Splicing; Reporter; Research; Research Personnel; Rheumatoid Arthritis; Role; Spinal cord injury; Tamoxifen; Testing; Therapeutic; Time; Tissues; Transgenic Mice; Transposition of Great Vessels; Variant; Wound Healing; base; carcinogenesis; cell behavior; cell type; chemokine; cytokine; homologous recombination; human disease; insight; mature animal; migration; monocyte; mouse model; promoter; public health relevance; recombinase; response; therapeutic development; tool; vector; versican

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.

描述（由申请人提供）：现在人们普遍认为细胞外基质（ECM）分子在健康和疾病中具有调节作用。尽管得到了这一认可，基因工程小鼠仍然无法用作研究大型硫酸软骨素蛋白多糖多功能蛋白聚糖（versican）的作用的工具，该蛋白聚糖与其结合伴侣透明质酸在多种急性和慢性炎症事件中都有涉及。我们的实验室一直参与动脉粥样硬化和肺部感染的研究，最近还合作研究哮喘和糖尿病，所有这些都具有关键的慢性免疫反应，其特征是多功能蛋白聚糖表达和积累增加。我们现在提议开发两种基因工程小鼠，这是确定多功能蛋白聚糖在健康和疾病中所扮演的角色的重要工具。在第一个目标中，我们将生成具有 versican 外显子的小鼠（versicanfl/fl 小鼠），这样当与表达可调节 Cre 重组酶的小鼠杂交时，可以通过同源重组实现 versican 表达的敲除（versican-/- 小鼠）。需要有条件的基因敲除，因为多功能蛋白聚糖基因的破坏会导致胚胎致死。在第二个目标中，我们将生成多功能蛋白聚糖报告基因小鼠模型，以确定控制多功能蛋白聚糖表达的机制。我们将生成一只带有多功能蛋白聚糖启动子的小鼠，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。然后，该靶向构建体将用于开发 versican 报告基因小鼠 (versicanTdTR/cspg2)。然后，我们将培育和表征 versicanTdTR/cspg2 小鼠，以表明 tdTomato 与 versican 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，目标 3 中拟议研究的成功完成将提供令人信服的证据，证明多功能蛋白聚糖在肺部感染的炎症反应中发挥着关键作用。一旦可用，这些基因工程小鼠将可用于研究多种与多功能蛋白聚糖相关的病理和损伤，例如脊髓损伤、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。明确确定多功能蛋白聚糖在特定炎症事件中的直接作用将刺激未来的治疗策略将多功能蛋白聚糖作为治疗慢性炎症的手段。 PHS 398（修订版。11/07）研究计划 公共健康相关性：该项目拟在小鼠中开发多功能蛋白聚糖基因的条件敲除，以及多功能蛋白聚糖报告小鼠，以用于未来的研究，以探索多功能蛋白聚糖作为与免疫细胞相互作用的细胞外基质的组成部分的作用。我们相信这些模型将具有广泛的用途，不仅可以获得对慢性炎症机制的新见解，还可以用于多功能蛋白聚糖被认为发挥作用的其他领域，例如脊髓损伤、癌症和出生缺陷。