Single-cell sampling and analysis

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Understanding the molecular mechanism underlying cellular function is a major goal in science. Unfortunately natural cell populations obtained from the blood or from various tissues are often difficult to study, as they can only be obtained in heterogeneous mixtures. For instance, stem cells found in adult tissues have a great potential in medicine for replacing damaged or diseased tissues. However, they are rare and cannot be harvested or grown to the large numbers needed for conventional bulk biochemical assays. Thus, single cell assays are essential for studying and understanding cell behaviour and the development of stem cell-based technologies and therapies. Capillary electrophoresis (CE), performed in fused-silica columns or on microchip channels, is a separation technology that is capable of high resolution separations of analytes based on size and charge from small volumes (< 10^-9 L). As such CE combined with the enhanced detection sensitivity of a laser-induced fluorescence detection scheme represents a powerful approach for the analysis of single mammalian cells. The overall objective of this proposal is to develop and optimize strategies for the biochemical analysis of single-cell by capillary and microchip electrophoresis. The first specific objective is to maximize the recovery of analytes from specific cell compartments by optimizing the method of cell sampling and the fabrication of the separation column. The second objective is to demonstrate the utility of single-cell CE to analyse posttranslational modifications of a membrane-tethered transcription factor important in stem cell fate decision, Notch. Single-cell electrophoresis in capillaries or on microchips is emerging as the next revolution in tools for biological discovery. Improved sampling and the ability to estimate losses will allow more sensitive and precise quantitation of fluorescent analytes in cells. Furthermore, once optimized, single-cell CE will provide a powerful means to study proteins that have complex posttranslational regulation such as the Notch protein. Consequently, single-cell CE has the potential to become an essential tool in development of drug, gene or cellular therapies targeting Notch and stem cells in general.