We have developed a high sensitivity capillary electrophoresis instrument that provides a measurement of protein expression in single somatic cells. We current resolve ~200 components, and anticipate improving the performance of the instrument to resolve over 1000 components. We have documented the performance of this instrument to study cell-cycle dependent protein expression in single HT29 colon cancer cells, the effect of apoptosis on protein expression in single MCF-7 breast cancer cells, and the effect of over expression of the transcription factor TWIST in single MC-3T3 osteoprecursor cells. While successful, the experiments are tedious, requiring roughly five hours to generate a two-dimensional electropherogram from a single cell. This proposal considers the development of high-throughput instrumentation for the generation of two-dimensional protein electropherograms from single cells. We will develop instruments to analyze 5-, 16-and 96-cells in parallel. The technology will be developed in an orderly progression, so that we will gain from the experience as we build progressively more complex instruments. Each instrument will incorporate a CCD camera to record an image of each cell before analysis. We will also develop cell-patterning technology to automatically inject cells into the instruments. We will evaluate the performance of the instruments. We will first use two capillaries to simultaneously aspirate two HT29 daughter cells immediately after mitosis. The protein electropherograms will reveal details on the cell-to-cell heterogeneity in protein packaging at mitosis. We will repeat the experiment to study daughter cells at varying times after mitosis to determine the dephasing of protein expression. We will study the effect of confluence on H9c2 cardiomyocite differentiation into myotubes. Cell patterning will be used to generate an array of cells with differing levels of confluence. Finally, we will monitor propagation of vesicular stomatitis virus and mouse hepatitis virus through cell cultures to study the systematic changes in protein expression following infection. Finally we will develop software tools to quantitate protein expression changes revealed by the single cell electropherograms. ? ?
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