eNOS: Metabolism & Vascular Biology in Health & Disease

描述（由申请人提供）： 拟议的研究有三个主要目标。首先（AIM 1）：开发过表达或低表达内皮型一氧化氮合酶（eNOS）的转基因猪（GMP）。 我们将通过向尤卡坦半岛胎儿成纤维细胞添加构建体（由内皮特异性Tie 2启动子、全长eNOS cDNA和选择性标记组成）来开发过表达eNO的猪，并使用核转移技术创建猪。这样的转基因动物将在内皮细胞中特异性地过表达eNOS。我们将通过在尤卡坦半岛胎儿成纤维细胞中同源重组，在外显子12中插入新霉素盒，开发缺乏eNOS（eNOS-/-）的猪。我们将使用用于核转移的靶向成纤维细胞产生杂合eNOS（+/-）猪，eNOS-/-猪将从eNOS猪的育种中产生，其在所有细胞类型中缺乏功能性eNOS蛋白。第二步（AIM 2）：确定这些GMP的代谢和心肺表型。 我们将描述GMP的全身代谢和心肺功能能力以及对冠状动脉血流的调节。从相同的动物，我们还将表征eNOS表达改变对骨骼肌和脂肪组织代谢以及血管内皮和平滑肌表型的影响。第三个目标（AIM 3）确定eNOS表达改变对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的影响。猪与人类共享肌肉和中间代谢以及心肺功能的重要特征，使它们成为我们对这些过程不断增长的理解的另一个重要环节。这些动物模型将成为公共和私营部门机构研究人员的有力工具，并将成为我们正在进行的关于eNOS在动脉粥样硬化血管疾病中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，以确定eNOS在以下方面的作用：1）肌肉和脂肪组织中代谢过程的控制（NIAMS），2）糖尿病和糖尿病血管疾病（NIDDK），以及3）生殖生物学（NICHD）。进一步开发用于生物医学研究的猪模型将减少使用非人灵长类动物模型进行涉及代谢和心肺系统遗传修饰的研究的压力。提交本申请的强大研究人员团队具有独特的资格，并准备完成这些目标。

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.