Genetic studies both at the organismal and cellular levels demand a sophisticated downstream analysis of the phenotypes that result from genetic manipulations. The creation of the transgenic and knockout animals proposed in this application, as well as the increasing capabilities of investigators using cell culture models provided by modern transient transfection technologies, will greatly increase the need for evaluating patterns of gene expression. Modern flow cytometry is the method of choice to answer these needs, especially because it provides unsurpassed capability to evaluate changes at the single cell level in large populations of cells. To accommodate the needs of this diverse group of investigators it is therefore proposed to establish a state of the art preparative flow cytometry core facility. In addition to all analytical procedures, this facility will have the ability to sort transiently transfected cells in a sterile fashion and provide investigators with pools of productively transfected viable cells that can be returned to culture for further observations or manipulation. This capability can in many cases obviate the need for stable transfection and thus result in a significant time saving. In addition, this method can be especially effective in establishing antisense ablation. The core will be supervised by Dr. John Sedivy, Professor, an expert in the field of cell cycle regulation, who has a long track record of publications using the proposed technologies. On a day to day basis the core will be run by a highly trained technician who will aid users in the preparation and analysis of their samples. Users of this core facility will have access not only to a state of the art flow cytometer, but also to guidance, expertise and training towards applying this technology to their own research needs.
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