Development of a novel genetic approach to study lung mesenchymal cell signaling

开发一种新的遗传方法来研究肺间充质细胞信号传导

• 批准号: 8176683
• 负责人: WEI SHI
• 金额: $ 24万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8176683
• 关键词: Age; Asthma; Bronchopulmonary Dysplasia; Cells; Cellular biology; Complex; DNA; Development; Dominant-Negative Mutation; Doxycycline; Elements; Enhancers; Epithelial Cells; Fetal Lung; Fluorescence; Functional disorder; Genes; Genetic; Growth Factor; Homeostasis; Knowledge; Lung; Lung diseases; Mesenchymal; Mesenchyme; Methodology; Mus; Pathogenesis; Pathway interactions; Play; Proteins; Publishing; Pulmonary Fibrosis; Regulatory Element; Reporter; Research; Role; Signal Pathway; Signal Transduction; Specificity; Staging; System; Testing; Tetanus Helper Peptide; Tetracyclines; Time; Trans-Activators; Transgenes; Transgenic Mice; Transgenic Organisms; dosage; in vivo; injury and repair; lung development; lung injury; lung maturation; mouse model; new therapeutic target; novel; postnatal; promoter; receptor; tool

DESCRIPTION (provided by applicant): Reciprocal interaction between lung mesenchymal and epithelial cells is essential for normal lung development and homeostasis. Therefore, alterations in lung mesenchymal cell biology will have a significant impact on both fetal lung formation and postnatal lung injury repair, resulting in the pathogenesis of many lung diseases. Although several sophisticated mouse genetic approaches have been obtained to study lung epithelial cell biology, the lack of lung mesenchymal cell-specific mouse genetic approaches to date has become a critical barrier to the field of lung research. We hypothesize that Tbx4 lung enhancer driven rtTA (reverse tetracycline- dependent transactivator) transgenic mouse line (Tbx4-rtTA) can be used to make a novel lung mesenchymal- specific Tet-On genetic system to study lung mesenchymal cell signaling in lung development and injury repair. Thus, combination of Tbx4-rtTA and TetO promoter driven dominant-negative TGF-¿ receptor II (dnT2RII) transgenic lines will provide a unique tool to study TGF-¿ signaling specifically in lung mesenchyme and its impact on lung development in vivo. Specific Aim 1. To develop and characterize a lung-mesenchymal cell-specific Tet-On genetic system. A 5.5 kb lung enhancer DNA element of Tbx4 gene will be used to make the Tbx4-rtTA transgenic mice. The lung mesenchymal localization of rtTA transgenic expression at different ages will then be verified. Furthermore, a lung mesenchymal cell-specific Tet-On genetic system will be generated and tested in mice by crossing Tbx4-rtTA and TetO-Cre mice with a floxed-mT-mG fluorescence protein reporter system. Specific Aim 2. To dissect the regulatory functions of TGF-¿ signaling in developing lung mesenchymal cells in vivo using the established lung mesenchymal Tet-On genetic approach. We will apply the newly developed lung mesenchymal Tet-On system to characterize TGF-¿ signaling in lung mesenchymal cells, as TGF- ¿ signaling plays extremely important roles in regulating lung development, injury repair, and lung diseases. By generating a Tbx4-rtTA/TetO-dnT2RII mouse line, lung mesenchymal-specific TGF-¿ signaling can be reversibly inhibited at various levels and stages in vivo through dnT2RII induction. The roles of mesenchymal TGF-¿ signaling in regulating lung development will then be dissected. Development of these novel genetic approaches has the potential to substantially extend current knowledge of lung mesenchymal cell biology and its relationship to lung diseases. Furthermore, by applying this system to lung mesenchymal TGF-¿ signaling research, we will be able to dissect the genetic dosages and time windows of TGF-¿ signaling in regulating lung development and maturation. Successful establishment of these unique genetic systems will provide powerful tools to define the role of mesenchymal signaling in lung development and homeostasis, and in the pathophysiology of lung diseases. Additionally, new therapeutic targets may be identified. PUBLIC HEALTH RELEVANCE: This proposed project will develop a novel genetic approach to specifically target lung mesenchymal cells in mouse model. Successful establishment of this methodology and its application to study TGF-¿ signaling in developing lung mesenchymal cells have the potential to substantially advance our research in lung mesenchymal cell biology. Moreover, these genetic tool and mouse models will help to break through the critical barrier in understanding the pathogenic mechanisms of several lung diseases including asthma and pulmonary fibrosis.

描述（由申请人提供）：肺间充质细胞和上皮细胞之间的相互作用对于正常肺发育和体内平衡至关重要。因此，肺间充质细胞生物学的改变将对胎儿肺形成和出生后肺损伤修复产生重大影响，导致许多肺部疾病的发病机制。尽管已经获得了几种复杂的小鼠遗传方法来研究肺上皮细胞生物学，但迄今为止缺乏肺间充质细胞特异性小鼠遗传方法已成为肺研究领域的关键障碍。我们假设Tbx 4肺增强子驱动的rtTA（反向四环素依赖性反式激活因子）转基因小鼠系（Tbx 4-rtTA）可用于制备新型肺间充质特异性Tet-On遗传系统以研究肺发育和损伤修复中的肺间充质细胞信号传导。因此，Tbx 4-rtTA和TetO启动子驱动的显性负性TGF-β受体II（dnT 2 RII）转基因系的组合将提供独特的工具来研究肺间充质中特异性的TGF-β信号传导及其对体内肺发育的影响。具体目标1。建立和鉴定肺间充质细胞特异性Tet-On遗传系统。将Tbx 4基因的5.5kb肺增强子DNA元件用于制备Tbx 4-rtTA转基因小鼠。然后将验证不同年龄rtTA转基因表达的肺间质定位。此外，将通过将Tbx 4-rtTA和TetO-Cre小鼠与标记的mT-mG荧光蛋白报告系统杂交，在小鼠中产生和测试肺间充质细胞特异性Tet-On遗传系统。具体目标2。使用已建立的肺间充质Tet-On遗传学方法，剖析TGF-β信号在体内发育的肺间充质细胞中的调节功能。我们将应用新开发的肺间充质Tet-On系统来表征肺间充质细胞中的TGF-<$信号传导，因为TGF-<$信号传导在调节肺发育、损伤修复和肺部疾病中起着极其重要的作用。通过产生Tbx 4-rtTA/TetO-dnT 2 RII小鼠系，肺间充质特异性TGF-β信号传导可以在体内通过dnT 2 RII诱导在不同水平和阶段被可逆地抑制。然后将剖析间充质TGF-β信号在调节肺发育中的作用。这些新的遗传学方法的发展有可能大大扩展目前对肺间充质细胞生物学及其与肺部疾病关系的认识。此外，通过将该系统应用于肺间质TGF-<$信号转导研究，我们将能够剖析TGF-<$信号转导在调节肺发育和成熟中的遗传剂量和时间窗。这些独特的遗传系统的成功建立将提供强有力的工具来定义间充质信号传导在肺发育和稳态中的作用，以及在肺部疾病的病理生理学中的作用。此外，还可以确定新的治疗靶点。 公共卫生相关性：该拟议项目将开发一种新的遗传方法，专门针对小鼠模型中的肺间充质细胞。这种方法的成功建立及其在发育中的肺间充质细胞中研究TGF-β信号传导的应用有可能大大推进我们在肺间充质细胞生物学方面的研究。此外，这些遗传工具和小鼠模型将有助于突破理解包括哮喘和肺纤维化在内的几种肺部疾病的致病机制的关键障碍。