Integrated Murine Models of COPD

2.目标 CORE C将为研究人员研究暴露在香烟烟雾(CS)和过滤空气(FA)中的WT和NULL小鼠的样本提供资源。为了实现这一目标，我们制定了以下五个主要目标。 2.1.目标1：提供在小鼠中建立COPD模型的专业知识：我们将为所有三个项目提供在MICE中建立COPD模型所需的专业知识和资源。这包括接触在CS暴露模型(欧文博士)、肺部炎症和损伤(欧文和佩雷拉博士)、成像系统(欧文博士)和组织病理学(佩雷拉博士)方面具有专业知识的个人。我们将为所有三个项目标准化CS诱导的COPD小鼠模型。在所有三个项目中，暴露于CS或FA的小鼠的肺组织都将用于功能目的。欧文博士一直是9楼肺科动物模型核心的主任 在BWH的荆棘楼工作了5年。这个核心已经让小鼠接触CS-10年了。欧文博士监督训练有素的技术人员执行和量化暴露，并监测暴露在CS中的小鼠。 通过使用同样训练有素和经验丰富的人员，我们将确保小鼠统一暴露于CS，暴露受到严格控制，并在整个过程中对小鼠进行适当监测。 2.2.目标2：培育和维护用于研究的新小鼠品系：佩雷拉博士已经培育了许多以基因为靶标的小鼠品系(18-23岁)，并将监督新小鼠品系的发展。我们要么已经获得了用于研究的小鼠(Ireb2-/-和HHIP+/-小鼠)，要么从KOMP储存库购买了转基因小鼠核心将用来产生HHIP和Fam13a基因靶向小鼠的ES细胞(见下文)。如果出现意想不到的问题，阻碍了在我们的CS暴露模型中对基因靶标小鼠的研究。佩雷拉博士非常有资格协助解决替代克隆策略的问题。在生成新行之后。欧文博士将监督 在我们的屏障设施中饲养小鼠，扩大研究群体，并对所有品系的小鼠进行基因分型，我们有充足的小鼠饲养空间(&gt；1000个笼子)。然后，欧文博士将协调将小鼠转移到烟雾暴露设施及其住所，然后进行CS和FA暴露(见3.1)。 2.3.目标3：组织采集、储存、分类和分配：欧文博士将指导训练有素的人员采集肺组织和血液，进行BAL，并分离和培养在气液界面(ALI)生长的肺泡巨噬细胞和原代肺上皮细胞。核心人员将与项目调查人员密切合作，以确保在CS或FA暴露后采集DNA、RNA、细胞和肺组织的项目之间的连续性。CORE C将存储、编目并将从小鼠身上分离的肺组织和原代细胞分发给所有三个项目，以确保有效利用暴露于CS的小鼠的组织。 2.4.目标4：组织病理学服务：佩雷拉博士自2001年以来一直指导这项服务，并指导一名经验丰富的组织学技术员(Bonna 1)。他将对组织进行石蜡包埋和切片，准备肺的冰冻切片，并对肺部进行常规组织化学染色和炎症标记染色。 该服务将根据需要对PPG人员执行的非常规染色进行故障排除。 2.5.目标5：成像和流式细胞术：这一核心组件包括一个明亮的视野和表观荧光显微镜、图像采集和分析软件、一个六色FACS Canto II流式细胞仪，以及可在Partners/HMS系统内使用共聚焦和电子显微镜核心以及细胞分类细胞仪。这个核心部分由欧文博士监督，她将协助所有使用这一资源的PPG调查人员。 2.6.总体目标：整合有技能的工作人员和设备，并将其提供给所有项目。核心C将降低制作高质量数据的成本，促进和促进不同项目之间的互动，并在成功完成本项目小组提出的工作方面发挥至关重要的作用。

2. Objectives Core C will provide resources for investigators studying samples from WT vs. null mice exposed to cigarette smoke (CS) vs. filtered air (FA). To accomplish this, we have developed five primary goals outlined below. 2.1. Goal 1: Provide Expertise in Modeling COPD in Mice: We will provide all three projects with the expertise and resources required to model COPD in mice. This includes access to individuals with expertise in CS exposure models (Dr. Owen), pulmonary inflammation and injury (Drs. Owen and Perrella), imaging systems (Dr. Owen), and histopathology (Dr. Perrella). We will standardize the murine model of CS-induced COPD for all three projects. Lung tissue from mice exposed to CS or FA will be used in functional aims in all three projects. Dr. Owen has been the Director of the Pulmonary Division Animal Models Core on the 9th floor of the Thorn Building at BWH for 5 years. This Core has been exposing mice to CS for -10 years. Dr. Owen supervises trained technicians to perform and quantify the exposures and to monitor mice being exposed to CS. By using the same highly trained and experienced personnel, we will ensure that mice are uniformly exposed to CS, exposures are tightly regulated, and mice are properly monitored throughout. 2.2. Goal 2: Development and Maintenance of Novel Lines of Mice for Study: Dr. Perrella has generated many gene-targeted lines of mice (18-23) and will oversee the development of new lines of mice. We have either already obtained mice for study (Ireb2-/- and Hhip+/- mice) or purchased ES cells transfected with gene-targeted constructs from the KOMP Repository which the HMS Transgenic Mouse Core will use to generate Hhip and Fam13a gene-targeted mice (vide infra). If unexpected problems arise that preclude study of the gene-targeted mice in our CS exposure model. Dr. Perrella is extremely well qualified to assist with troubleshooting of alternative cloning strategies. After new lines are generated. Dr. Owen will oversee the murine husbandry, expansion of colonies for study, and genotyping of all lines of mice in our barrier facility where we have ample murine housing space (>1000 cages). Dr. Owen will then coordinate transfers of the mice to the smoke exposure facility and their housing and CS vs. FA exposures thereafter (see 3.1). 2.3. Goal 3: Tissue Harvesting, Storage, Cataloging, and Distribution: Dr. Owen will supervise highly trained personnel to harvest lung tissue and blood, perform BAL, and isolate and culture alveolar macrophages and primary lung epithelial cells grown at an air-liquid interface (ALI). Core personnel will work closely with project investigators to ensure continuity among the projects for harvesting of DNA, RNA, cells, and lung tissue after CS or FA exposures. Core C will store, catalog, and distribute lung tissue and primary cells isolated from mice to all three projects to ensure efficient usage of tissues from mice exposed to CS. 2.4. Goal 4: Histopathology Service: Dr. Perrella has directed this service since 2001 and supervises an experienced histology technician (Bonna 1th). He will paraffin-embed and section tissues, prepare frozen sections of lungs, and perform routine histochemical staining and staining of lungs for markers of inflammation. This service will troubleshoot non-routine staining performed by PPG personnel as needed. 2.5. Goal 5: Imaging and Flow Cytometry: This Core component contains a bright field and epi-fluorescence microscope, image acquisition and analysis software, a six-color FACS Canto II flow cytometer, and access to Confocal and Electron Microscopy Cores and cell sorting cytometers within the Partners/HMS system. This Core component is overseen by Dr. Owen, and she will assist all of the PPG investigators using this resource. 2.6. Overall goals: By consolidating skilled staff and equipment and making them available to all projects. Core C will reduce the cost of producing high-quality data, facilitate and promote interactions between the different projects, and play a critically Important role in the successful completion of work proposed in this PPG.