Core: Molecular biology

改编自申请者？S摘要)所有提出的个人项目 在这个整体计划项目中，结合了分子生物学的使用 回答各种不同的科学问题的技巧 生理模型。分子生物学核心设施将提供 实施这些技术的资源和专业知识。分子生物学 实验室和组织培养设施建立在 生理学，1994年2月。分子生物学实验室很好 装备齐全。它是一个独立的实验室空间，其中包含标准设备，如 作为索瓦尔离心机，分光光度计，荧光计，两种杂交 烤箱，一台Eppendorf聚合酶链式反应机器，一台genmyxLR DNA测序仪，一个细菌摇床 和保育箱，以及足够的工作台空间，水浴等。此外 设置了凝胶扫描仪和摄像机，用于图像记录和 对实验进行了量化分析。组织培养设施包括两个 生物安全柜和两个二氧化碳孵化器，其中一个具有 控制氧气水平，以及带摄像机的相差显微镜 连接到成像系统的附件。组织培养设施是一个 生物安全2级设施与腺病毒繁殖的修改。 因此，它适合于人类细胞系的培养和生产。 重组腺病毒和逆转录病毒。到目前为止，许多技术已经被 由该司的成员执行。有几个细胞系通常是 原代培养的肺胎儿成纤维细胞和L8骨骼肌 细胞以及病毒包装细胞系。细胞增殖分析是 通过荧光法测量总DNA和DNA合成来实现 3h胸腺嘧啶核苷掺入。目前在RNA水平上的基因表达 通过Northern分析和定量RT-PCR方法对 小样本量，如人体活检、颈动脉体样和单个 肌肉纤维。该实验室的建立也是为了测量转录 通过核连续分析和瞬时转染法测定其活性。在 蛋白质水平的蛋白质水平分析是常规执行的一些 蛋白质、组织和前胶原的合成目前是用 细菌胶原酶消化试验，尽管在DR中有BPLC可用。 鲍威尔？S实验室分离羟基脯氨酸和游离脯氨酸 放射性脉冲肺组织。正在进行中，与 形态/形态测量核心设备，以及原位杂交分析 因为同时使用颜色和荧光检测系统的免疫组织化学 已执行。细菌细胞进行常规培养以进行亚克隆、分离 用于Northern分析的探针的cDNA质粒的纯化，原位 杂交技术以及有条件的基因敲除实验 正在进行中。最后，差异显示技术和DNA 序列分析用于鉴定新的交替表达的mRNA物种 组织和细胞实验正在进行中。实验室已经培训了 该司在这些技术方面的几名成员，包括技术人员， 研究生、博士后研究员和访问科学家。其核心是 该机构由Ellen Breen博士指导，他在 分子生物学技术。下面列出了以下项目的表格 将利用分子生物学核心设施和主要技术 这将会被雇佣。

Adapted from the Applicant?s Abstract) All of the individual projects proposed in this overall program project incorporate the use of molecular biology techniques to answer specific scientific questions addressed in various physiological models. A molecular biology core facility will provide the resources and expertise to carry out these techniques. A molecular biology laboratory and tissue culture facility were established in the Division of Physiology in February 1994. The molecular biology laboratory is well equipped. It is a separate laboratory space containing standard equipment such as a Sorvall centrifuge, spectrophotometer, fluorometer, two hybridization ovens, an Eppendorf PCR machine, a genomyxLR DNA sequencer, a bacterial shaker and incubator as well as adequate bench space, water baths etc. In addition a gel scanner and video camera are set up for image documentation and quantitative analysis of experiments. The tissue culture facility contains two biosafety cabinets, and two CO2 incubators one of which has the capability of controlling O2 levels, and a phase contrast microscope with video camera attachment connected to the imaging system. The tissue culture facility is a biosafety 2 level facility with modification for adenovirus propagation. Therefore, it is appropriate for culture of human cell lines and production of recombinant adenoviruses and retroviruses. To date many techniques have been carried out by members of the Division. Several cell lines are routinely cultured including primary pulmonary fetal fibroblasts and L8 skeletal muscle cells as well as viral packaging cell lines. Cell proliferation assays are accomplished through measurement of total DNA by fluorometry and DNA synthesis by 3h thymidine incorporation. Gene expression at the RNA level is currently analyzed by Northern analysis and quantitative RT-PCR methods are used for small samples sizes such as human biopsies, carotid body samples and single muscle fibers. The laboratory is also set up to measure transcriptional activity by nuclear run-on analysis and transient transfection assay. At the protein level Western analyses are routinely performed for a number of proteins and tissues and procoliagen synthesis is currently measured using the bacterial collagenase digestion assay although an BPLC is available in Dr. Powell?s laboratory for the separation of hydroxyproline and proline in radioactively pulsed lung tissue. Ongoing, in conjunction with the morphology/morphometry core facility, in situ hybridization analyses as well as immunohistochemistry using both color and fluorescent detection systems are performed. Bacterial cells are routinely cultured for subcloning, isolation and purification of CDNA plasmids used as probes in northern analyses, in situ hybridization techniques as well as conditional knock out experiments which are under way. Finally, the technique of differential display and DNA sequence analysis to identify new mRNA species alternatively expressed in tissue and cell experiments is in progress. The laboratory has trained several members of the division in these techniques including technical staff, graduate students, postdoctoral fellows and visiting scientists. The core facility is directed by Dr. Ellen Breen who has extensive experience in molecular biology techniques. Listed below is a table of the projects which will make use of the Molecular Biology Core Facility and the main techniques that will be employed.