eNOS: Metabolism & Vascular Biology in Health & Disease

描述(由申请人提供)： 这项拟议的研究有三个主要目标。第一(目标1)；培育高表达或低表达内皮型一氧化氮合酶(ENOS)的转基因猪(GMP)。我们将通过在尤卡坦胎儿成纤维细胞中添加一个构建体(由内皮特异性Tie-2启动子、全长eNOS cDNA和一个可选择的标记组成)来培育过表达eNO的猪，并使用核移植技术创造猪。这样的转基因动物将在内皮细胞中特异地过度表达eNOS。我们将在尤卡坦的胎儿成纤维细胞中，通过同源重组，用新霉素盒替换第12外显子，培育出缺乏eNOS(eNOS-/-)的猪。我们将使用靶向成纤维细胞来培育杂合型eNOS(+/-)猪进行核移植，eNOS-/-猪将从eNOS猪的繁育中产生，这些猪在所有细胞类型中都缺乏功能性eNOS蛋白。第二(目标2)；建立这些GMP的代谢和心肺表型。我们将研究GMPs的全身代谢和心肺功能，以及冠脉血流的调节。我们还将从相同的动物身上表征eNOS表达变化对骨骼肌和脂肪组织代谢的影响，以及血管内皮细胞和平滑肌的表型。最后(目的3)；确定eNOS表达改变在高脂饮食诱导的高脂血症引起的内皮功能障碍和血管疾病中的作用。猪与人类分享肌肉和中间代谢以及心肺功能的重要特征，使它们成为我们日益了解这些过程的另一个重要环节。这些动物模型将成为公共和私营机构研究人员的强大工具，并将成为我们正在进行的关于eNOS在动脉粥样硬化性血管疾病中的作用的研究的重要工具(由NHLBI支持)。此外，这些GMP将提供重要的动物模型来确定eNOS在以下方面的作用：1)肌肉和脂肪组织代谢过程的控制(NIAMS)；2)糖尿病和糖尿病血管疾病(NIDDK)；3)生殖生物学(NICHD)。用于生物医学研究的猪模型的进一步发展将减少使用非人类灵长类动物模型进行研究的日益增长的压力，这些研究涉及代谢和心肺系统的基因修改。提交这份申请的强大的调查小组是唯一有资格和准备实现这些目标的。

DESCRIPTION (provided by applicant): The proposed research has three major goals. First (AIM 1); develop genetically modified pigs (GMPs) that overexpress or under-express endothelial nitric oxide synthase (eNOS). We will develop pigs that over-express eNO by adding a construct (composed of an endothelial-specific Tie 2-promoter, full length eNOS cDNA and a selectable marker) to Yucatan fetal fibroblast cells and create the pigs using nuclear transfer technology. Such a transgenic animal will over express eNOS specifically in endothelial cells. We will develop pigs that lack eNOS (eNOS-/-) by replacin exon 12 with a neomycin cassette by homologous recombination in Yucatan fetal fibroblast cells. We will produce heterozygous eNOS (+/-) pigs using targeted fibroblast cells for nuclear transfer, eNOS-/- pigs, that lack functional eNOS protein in all cell types, will be generated from the breeding of eNOS pigs. Second (AIM 2); establish the metabolic and cardiorespiratory phenotype of these GMPs. We will characterize whole body metabolism ana cardiorespiratory functional capacities of GMPs and the regulation of coronary blood flow. From the same animals we will also characterize the effects of altered eNOS expression on metabolism of skeletal muscle and fat tissues, ana thephenotype of vascular endothelium and smooth muscle. Finally (AIM 3); determine the effects of altered eNOS expression on endothelial dysfunction and vascular disease produced by high fat diet-induced hyperlipidemia. Pigs share with humans important characteristics of muscle and intermediary metabolism and of cardiorespiratory function, making them another important link in our growing understanding of these processes. These animal models will be powerful tools for investigators at both public and private sector institutions and will form an important tool for our ongoing research concerning the role of eNOS in atherosclerotic vascular disease (supported by NHLBI). In addition, these GMPs will provide important animal models to determine the role of eNOS in: 1) the control of metabolic processes in muscle and fat tissue (NIAMS), 2) diabetes and diabetic vascular disease (NIDDK), and 3) reproductive biology (NICHD). Further development of porcine models for biomedical research will reduce the growing pressure Ion the use of nonhuman primate models for research involving genetic modifications of metabolic and cardiorespiratory systems. The strong group of investigators submitting this application is uniquely qualified and prepared to accomplish these goals.