Molecular and Protein Core

分子生物学，在广义上涵盖了DMA和RNA的操作，已经成为一种 大多数生物研究项目不可或缺的一部分，包括北卡罗来纳大学项目资助(PPG)。 利用分子生物学技术的研究领域包括克隆表达蛋白质的基因 细胞模型系统，用于细胞定位和功能研究的定点突变和标记， 聚合酶链式反应用于克隆和基因分型，RNA分离和表达分析，以及 转基因小鼠的发展。分子生物学核心在诺斯大学成立 卡罗莱纳(北卡罗来纳州)囊性纤维化中心于1998年为教职员工提供分子生物学服务和专业知识 他们通常主要接受细胞生物学和/或生理学方面的培训。核心目前是 正在进行的PPG主要提供克隆和抗体生产服务。核心的功能有 在最新的提案中进行了扩展，以适应新项目的需要，现在包括开发和 转基因小鼠模型的基因分型和RNA表达分析服务。具体地说，PPG 研究人员将要求构建腺病毒、逆转录病毒和非洲爪哇表达载体 表达各种标记和/或修饰的蛋白质，如CFTR、ENaC亚基、蛋白酶和 多种细胞模型中的核苷酸转运蛋白，包括分化良好的人支气管上皮 (HBe)细胞培养(由Core C.细胞培养提供)。此外，分子生物学/小鼠基因分型 CORE将提供新的转基因小鼠模型(包括敲除和过度表达 模型)，以研究核苷酸和核苷在呼吸道表面液体中释放和代谢的作用 并研究蛋白水解酶在ENaC功能调节中的作用。与此同时，基因分型 用于产生实验动物和对照动物的转基因模型也将由 分子生物学/小鼠基因分型核心。最后，RNA分离和表达分析服务， 包括获得定量实时聚合酶链式反应设备和专业知识。此外， CORE正在继续努力提供获得新研究工具的途径，包括开发siRNA 技术，我们将寻求将这些技术作为协议和协议提供给PPG调查人员 制定了应对策略。分子生物学/小鼠基因分型核心将被所有四个人利用 拟议的PPG项目，因此是本申请书中概述的努力的一个组成部分。

Molecular biology, which in a broad sense covers the manipulation of DMA and RNA, has become an integral part of most biological research programs, including the UNC Program Project Grant (PPG). Research areas that utilize molecular biology techniques include cloning genes for expression of proteins in cell model systems, site-directed mutagenesis and tagging for cellular localization and functional studies, polymerase chain reaction for cloning and genotyping, RNA isolation and expression analysis, and the development of transgenic mice. The Molecular Biology Core was established at the University of North Carolina (UNC) Cystic Fibrosis Center in 1998 to provide molecular biology services and expertise to faculty who are often primarily trained in cell biology and/or physiology. The Core is currently an integral part of the ongoing PPG to provide primarily cloning and antibody production services. The functions of the Core have been expanded in the latest proposal to suit new project needs, and now include the development and genotyping of transgenic mouse models and RNA expression analyses services. Specifically, the PPG investigators will require the construction of adenoviral, retroviral, and Xenopus expression vectors for expression of a variety of tagged and/or modified proteins, such as CFTR, ENaC subunits, proteases, and nucleotide transporters, in a variety of cell models including well-differentiated human bronchial epithelial (HBE) cell cultures (provided by Core C. Cell Culture). In addition, the Molecular Biology/Mouse Genotyping Core will provide for the generation of new transgenic mouse models (both knock-out and overexpressing models) to study the role of nucleotides and nucleosides release and metabolism in the airway surface liquid regulation and to study the role of proteases in the regulation of ENaC function. Along with this, genotyping of transgenic models for generation of experimental and control animals will also be provided by the Molecular Biology/Mouse Genotyping Core. And finally, RNA isolation and expression analysis services, including access to quantitative real-time PCR equipment and expertise, will be provided. In addition, the Core is continuously striving to provide access to new research tools, including the development of siRNA technologies, and we will seek to make these technologies available for PPG investigators as protocols and strategies are developed. The Molecular Biology/Mouse Genotyping Core will be utilized by all four proposed PPG projects, and thus is an integral component of the efforts outlined in this application.