Core: Molecular biology

核心：分子生物学

• 批准号: 6589835
• 负责人: Ellen C Breen
• 金额: $ 24.62万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6589835
• 关键词: biomedical facility; immunocytochemistry; in situ hybridization; molecular biology; northern blottings; nuclear runoff assay; nucleic acid sequence; tissue /cell culture; transfection; western blottings

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.

是否改编自申请人？s摘要）所有提出的个别项目 在这个整体计划项目中， 技术来回答各种不同的科学问题， 生理模型分子生物学核心设施将提供 资源和专业知识来执行这些技术。分子生物学 实验室和组织培养设施已建立在司 1994年2月的生理学。分子生物学实验室 搭载这是一个独立的实验室空间，包含标准设备， 如Sorvall离心机、分光光度计、荧光计、二次杂交 烘箱、Eppendorf PCR仪、genomyxLR DNA测序仪、细菌摇床 和孵化器以及足够的工作台空间，水浴等。此外， 设置凝胶扫描仪和摄像机用于图像记录， 实验的定量分析。组织培养设施包含两个 生物安全柜和两个二氧化碳培养箱，其中一个具有以下能力： 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，经改良用于腺病毒增殖。 因此，其适用于人细胞系的培养和人细胞的生产。 重组腺病毒和逆转录病毒。到目前为止，已经有许多技术 由分部成员执行。几种细胞系常规地 培养包括原代肺胎儿成纤维细胞和L 8骨骼肌 细胞以及病毒包装细胞系。细胞增殖测定是 通过荧光法和DNA合成法测量总DNA完成 3 h胸腺嘧啶核苷掺入法。目前，RNA水平的基因表达 通过北方分析和定量RT-PCR方法进行分析， 小样本量，如人体活检、颈动脉体样本和单个 肌肉纤维该实验室还建立了测量转录 活性通过核连续分析和瞬时转染测定。在 蛋白质水平Western分析常规地对许多 蛋白质和组织以及前胶原的合成目前是使用 细菌胶原酶消化试验，尽管BPLC在Dr. 鲍威尔的实验室分离羟脯氨酸和脯氨酸， 放射性脉冲肺组织。正在进行，与 形态学/形态测量学核心设施，原位杂交分析以及 由于使用颜色和荧光检测系统的免疫组织化学， 执行。常规培养细菌细胞用于亚克隆、分离 在原位北方分析中用作探针的cDNA质粒的纯化 杂交技术以及条件敲除实验， 正在进行中。最后，差异显示技术和DNA 序列分析，以确定新的mRNA物种交替表达， 组织和细胞实验正在进行中。实验室培训了 该司的几名成员，包括技术人员， 研究生、博士后和访问科学家。核心 该设施由艾伦·布林博士指导，他在以下方面拥有丰富的经验： 分子生物学技术以下是一个项目表， 将利用分子生物学核心设施和主要技术 将被雇用。