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.
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