CORE--CELL BIOLOGY SUPPORT

核心--细胞生物学支持

• 批准号: 6602444
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6602444
• 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 具有多种特征良好的培养细胞的项目 来自血管和非血管来源。核心人员还拥有 根据需要与研究人员密切合作开发新的培养系统 促进该计划的科学目标的实现。在上一个项目期间 在此期间，这包括开发两种新的方法 使用 FACS 分选和抗体分离小鼠肺内皮 耦合磁珠。目前正在进行扩大工作 应用这些方法分离鼠内皮细胞 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，这将是一个主要优先事项，并且应该 允许研究人员检查这些之间潜在的重要差异 从正常和遗传中分离出的各种类型的内皮细胞 改造动物。 核心单位 A 将继续努力尽量减少重复成本 在该计划内并提供关键的质量控制测试 生物试剂。胎牛血清、生长和附着因子、 核心人员定期测试酶的功效，并且 然后批量购买以供项目研究人员使用。这 确保核心中衍生的细胞培养物维持在原样 从最初的孤立到 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培训 项目人员（例如技术人员、学生、研究员）。核 人员还将对新人员进行技术和程序方面的培训 安全处理、净化和处置所必需的 生物危害材料，例如人体组织、人体血液和病毒 被感染的细胞。 在下一个项目期间，Core I将承担两项新功能。 一、胚胎干细胞系的维持及附加细胞 将携带小鼠“基因打靶”所需的培养材料 由核心A人员出炉。在上一个项目期间， 转基因/ES细胞核心产生的小鼠携带多种新突变 内皮细胞粘附分子，从而获得专业知识和 展示了在基因操纵方面的良好记录 体外和体内小鼠基因组 (1-3)。在更新期间， 该领域的专业知识将被纳入细胞生物学核心。作为 如下所述，Core A人员将协助维护ES 细胞系，而实际的囊胚注射将在 LMRC 机构转基因核心设施（在 阿琳·夏普博士。其次，核心将维护和监督 一种新的荧光显微镜/计算机图像分析系统和 现有的荧光分光光度/显微镜系统 被项目 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能够接触到最先进的技术 定量荧光显微镜、图像分析和出版 优质色彩和 B/2 数字印刷。现有SPEX双励磁 分光光度计将允许一般荧光测定和生存时间 细胞内 Ca2+ 浓度的荧光显微镜分析 使用 Fura-2 和新一代非紫外荧光探针 测量胞质单价或二价阳离子和 pH 值。博士。 Luscinskas 和 Edward Marcus 先生将提供协助 研究人员计划和执行此类实验。