CORE--CELL BIOLOGY LABORATORY

细胞生物学核心将提供培养的细胞，用于确定 转染后细胞的活性和功能，以及 研究转基因造血干细胞所需的技术。 表面和腺上皮细胞的高分化原代培养 来自人的气管(包括CF和非CF)将在DR中启动。 Finkbeiner的实验室，并在Widdicombe博士的实验室维护。 气道腺细胞培养最初是在加州大学旧金山分校开发的，现在还没有 在其他机构普遍提供。越来越多的证据表明 腺体分泌异常在CF的呼吸道病理中起重要作用 增强了这些细胞的价值。转化的呼吸道上皮细胞 将提供管路(腺体和表面上皮、CF和非CF) 克鲁内特博士写的。其他细胞系将在 根据需要进行培养。目前有各种各样的技术 在Widdicombe博士的实验室建立，用于评估氯的分泌 细胞培养的功能。其中包括Ussing暗室、放射性示踪剂 通量、SPQ荧光测量和膜片钳。此外 为了运输化验，其他细胞生物技术将是可用的， 包括活力测试，细胞分离，蛋白质检测 Western blotting，[Ca~(2+)]i和cAMP测定， 蛋白激酶活性和蛋白磷酸化。其有效性 在实验动物和人类中体内转导CFTR的研究将是 通过测量跨上皮气道势差进行评估 (P.D.)(在适当的条件下，本警署。是营地的一种衡量标准- 依赖的氯分泌)。在帕拉维奇尼博士的实验室里，专业知识是 可用于a)从以下来源浓缩小鼠或人类造血干细胞 骨髓标本(转基因后)，b)移植小鼠干细胞 和/或整个骨髓移植到小鼠受体中，以及c)评估干细胞和 人和小鼠造血组织中的祖细胞频率。这个 携带转基因的造血细胞的频率将是 用集落试验和整体评估造血祖细胞 纸巾。转基因基因的表达将用 分子/生化分析(由每名研究人员提供)。 将使用分子分析来监测捐赠者细胞的植入 基因标记的供体细胞。转基因DNA的整合 序列将使用荧光原位杂交进行测量。

The Cell Biology CORE will provide cultured cells, assays for determining the viability and function of cells following transfection, and the technology needed for studies of transfected hemopoietic stem cells. Highly differentiated primary cultures of surface and gland epithelium from human trachea (both CF and non-CF) will be initiated in Dr. Finkbeiner's laboratory, and maintained in Dr. Widdicombe's laboratory. Airway gland cell cultures were initially developed at UCSF, and are not generally available at other institutions. Increasing evidence that abnormal gland secretion contributes to the airway pathology of CF greatly enhances the value of these cells. Transformed airway epithelial cell lines (gland and surface epithelium, CF and non-CF) will be made available by Dr. Cruenert. Other cell lines will be initiated and maintained in culture as required. A wide variety of techniques are currently established in Dr. Widdicombe's laboratory for assessing the Cl secretory function of cell -cultures. These include Ussing chambers, radiotracer fluxes, measurements of SPQ fluorescence, and patch-clamping. In addition to transport assays, other cell biological techniques will be available, including tests of viability, cell fractionation, detection of proteins by Western blotting, measurement of [Ca2+]i and cAMP, determination of protein kinase activities and protein phosphorylation. The effectiveness of in vivo CFTR transfection in experimental animals and humans will be assessed from measurements of transepithelial airway potential difference (p.d.) (under the appropriate conditions, this p.d. is a measure of cAMP- dependent Cl secretion). In Dr. Pallavicini's laboratory, expertise is available to a) enrich either murine or human hemopoietic stem cells from marrow specimens (after transfection), b) transplant murine stem cells and/or whole marrow into murine recipients, and c) assess stem and progenitor frequencies in both human and mouse hemopoietic tissues. The frequency of hemopoietic cells carrying the transfected gene will be assessed in hemopoietic progenitors using colony assays and in whole tissues. Expression of transfected genes will be determined using molecular/biochemical assays (to be provided by each investigator). Engraftment of donor cells will be monitored using molecular analysis of genetically tagged donor cells. Integration of the transfected DNA sequences will be measured using fluorescence in situ hybridization.