CORE--CELL BIOLOGY SUPPORT

核心--细胞生物学支持

• 批准号: 6469270
• 负责人: FRANCIS W. LUSCINSKAS
• 金额: $ 6.87万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6469270
• 关键词: animal tissue; biomedical facility; cell line; flow cytometry; fluorescence microscopy; gene targeting; human tissue; tissue /cell culture; vascular endothelium

Since its inception in 1978, this core has provided researchers within the Program Project with a wide variety of well-characterized cultured cells from both vascular and non-vascular sources. Core personnel also have worked closely with researchers to develop new culture systems, as needed to facilitate the scientific goals of the Program. During the last project period, this has included the development of two new methodologies for the isolation of mouse lung endothelium using FACS sorting and antibody- coupled magnetic beads. Work is currently underway to expand the application of these methodologies to isolate murine endothelial cells from different microvascular beds, including the heart, kidney and brain. During the next project period, this will be a major priority, and should allow researchers to examine potentially important differences among these various types of endothelium isolated from both normal and genetically modified animals. Core Unit A will continue in its efforts to minimize cost duplication within the Program and to provide quality control testing of key biological reagents. Fetal bovine serum, growth and attachment factors, and enzymes are routinely tested for their efficacy by Core personnel, and then purchased in bulk quantities for use by Program researchers. This ensures that cell cultures derived in the Core are maintained in as consistent a fashion as possible from their initial isolated through to their experimental uses in the Projects. The Core will continue to provide training in general cell culture methods and in sterile technique to new Program personnel (e.g., technicians, students, research fellows). Core personnel also will train new personnel in the techniques and procedures necessary for the safe handling, decontamination, and disposal of biohazardous materials such as human tissue, human blood, and virally infected cells. During the next project period, Core I will assume two new functions. First, the maintenance of embryonic stem cell lines and additional cell culture materials required for "gene targeting" in mice will be carried out by Core A personnel. During the previous project period, the Transgenic/ES Cell Core produced mice bearing several new mutations in endothelial cell adhesion molecules, thus gaining expertise and demonstrating a proven track record in the genetic manipulation of the murine genome both in vitro and in vivo (1-3). In the renewal period, expertise in this area will be incorporated into the Cell Biology Core. As described below, Core A personnel will assist in the maintenance of ES cell lines, while the actual blastocyst injections will be carried out in the LMRC Institutional Transgenic Core facility (under the supervision of Dr. Arlene Sharpe. Second, the Core will maintain and oversee the use of a new fluorescence microscopy/computerized image analysis system and an existing fluorescent spectrophotometric/microscopy system which will be heavily utilized by Projects 1, 2, 3, and 4. The new computer imaging system will give Project researchers access to state-of-the-art quantitative fluorescence microscopy, image analysis and publication quality color and B/2 digital printing. The existing SPEX dual excitation spectrophotometer will allow general fluorimetry and live time fluorescence microscopic analysis of intracellular Ca2+ concentrations using Fura-2 and newer generation non-UV fluorescent probes for measurement of cytosolic monovalent or divalent cations and pH. Dr. Luscinskas and Mr. Edward Marcus will be made available to assist researchers in planning and executing such experiments.

自1978年成立以来，该核心为研究人员提供了 计划项目具有各种特征良好的培养细胞 来自血管和非血管来源。核心人员也有 与研究人员紧密合作，根据需要开发新的文化系统 促进该计划的科学目标。在最后一个项目中 时期，这包括开发两种新方法 使用FACS分类和抗体分离小鼠肺内皮 耦合磁珠。目前正在进行工作以扩展 这些方法的应用以分离鼠内皮细胞 来自不同的微血管床，包括心脏，肾脏和大脑。 在下一个项目期间，这将是重中之重，应该 允许研究人员研究其中的潜在重要差异 从正常和遗传上分离的各种类型的内皮 改良的动物。 核心单位A将继续努力，以最大程度地减少成本重复 在程序中并提供密钥的质量控制测试 生物试剂。胎牛血清，生长和依恋因子， 并经常通过核心人员对酶进行疗效进行测试，并且 然后，批量购买供计划研究人员使用。这 确保在核心中得出的细胞培养物保持在AS中 从最初隔离到 他们在项目中的实验用途。核心将继续提供 一般细胞培养方法和无菌技术的培训 计划人员（例如技术人员，学生，研究研究员）。核 人员还将在技术和程序中培训新人员 安全处理，净化和处置所需的必要 生物危害材料，例如人体组织，人类血液和病毒 感染细胞。 在下一个项目期间，核心我将采用两个新功能。 首先，维持胚胎干细胞系和其他细胞 将携带“基因靶向”所需的培养材料 由核心人员出发。在上一个项目期间， 转基因/ES细胞核产生的小鼠含有几个新突变 内皮细胞粘附分子，从而获得专业知识和 在遗传操作中证明了良好的记录 体内和体内的鼠基因组均在体外（1-3）。在续签时期， 该领域的专业知识将纳入细胞生物学核心。作为 下面描述的是，核心人员将有助于维护ES 细胞系，而实际的胚泡注射将在 LMRC机构转基因核心设施（在 Arlene Sharpe博士。其次，核心将维护和监督使用 新的荧光显微镜/计算机图像分析系统和一个 现有的荧光分光光度计/显微镜系统将是 项目1、2、3和4大量使用。新的计算机成像 系统将使项目研究人员访问最先进 定量荧光显微镜，图像分析和出版物 优质的颜色和B/2数字印刷。现有的SPEX双重激发 分光光度计将允许一般的荧光学和实时时间 细胞内Ca2+浓度的荧光显微镜分析 使用Fura-2和较新的非UV荧光探针 胞质单价或二价阳离子和pH的测量。博士 Luscinskas和Edward Marcus先生将提供帮助 计划和执行此类实验的研究人员。