Acquisition of a FACSort for Studying Lymphocyte Differentiation and for Undergraduate Research Training

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ABSTRACT We are requesting funding for a Becton Dickinson FACsort. There are three main objectives for the acquisition of this instrument. 1) The FACsort will be used extensively in ongoing studies on lymphocyte differentiation. These projects involve 6 to 8 undergraduate students every term, including the summer. 2) The FACSort will be an integral part of the undergraduate Immunology course. 3) Experiments using the FACSort will be introduced into the Molecular Biology course. Thus, a large number of undergraduates will gain experience with the fluorescence activated cell sorting through both undergraduate courses and research experience. Two research projects at Occidental College require cell sorting. Dr. Pollock's research centers on somatic mutation of immunoglobulin (Ig) variable (V) region genes. Somatic mutation plays an important role in generating antibody diversity. We are developing an assay to detect and isolate lymphocytes undergoing somatic mutation of immunoglobulin genes. The assay uses a DNA construct encoding a fusion protein composed of a rearranged V gene, containing a stop codon, linked to a lacZ reporter gene. If somatic mutation reverts the stop codon, the entire fusion protein is translated and lacZ expressed. LacZ expression is readily detected by fluorescence assays. This construct is being used in cell lines and to make transgenic mice. These mice will be used to ask: 1) where can somatic mutation be detected; 2) when is somatic mutation seen after immunization; 3) when does somatic mutation first occur during ontogeny; 4) which germinal center cells show active somatic mutation; and 5) what is the phenotype of cells undergoing somatic mutation.