CORE--CELL BIOLOGY SUPPORT

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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.