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Isolating a Cell Maximally Secreting Acetylcholinesterase

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Annual rept. 31 Jan 1984-30 Jan 1985

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Basic methods were developed for the isolation of subpopulations of cells which are high producers of a desired secreted cell protein. The ultimate goal of this research is to isolate a cell line maximally secreting human acetylcholinesterase AChE, acetylcholine hydrolase. This positive selection system, termed the Cell Isolation Technique CIT, has evolved in two different directions to screen individual cells trapped within agarose beads. Both approaches rely on a specific ligand-receptor antigen-antibody interaction to capture the desired secreted protein and immobilize it within the beads. In both cases, the cells secrete product, product binds to immobilized specific reagents and thus accumulates within the beads. Beads with high densities of desired product are identified and physically isolated so as to enrich for subpopulations of high producer cells. The two approaches differ with respect to how they identify and sort the beads with desired cells. In the original method, simple density gradient centrifugation is sufficient to separate beads with lysed red blood cells from the vast majority of beads with intact cells. The second approach uses a fluorescence activated cell sorter to screen beads which have accumulated captured secreted cell product now identified with fluorescently labeled antibodies. Although a cell line secreting high levels of AChE has not yet been developed by these methods, model studies have been encouraging. This report will therefore focus on the basic research behind this technology and its application for screening cells transfected with total genomic human DNA. Keywords Rhabdomyosarcoma cells.

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  • Biochemistry
  • Genetic Engineering and Molecular Biology

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