This core unit will provide sophisticated three color immunofluorescent cell analysis and three color cell sorting by providing a dedicated fluorescence activated cell sorter, and full time technical operator, to support the five collaborative research projects which form the basis of this research program project proposal. Each of the collaborative projects in this program rely heavily on the use of fluorescence activated cell analysis and cell sorting to achieve their experimental goals. Thus, Dr. Chien utilizing data on dominant peptide epitopes provided by Dr. McDevitt, will develop techniques for production of polyvalent specific peptide/I-/A/g7 staining reagents which can detect all of the various T cells specific for a particular peptide/I-A/g7 complex appearing in the course of the development of IDDM in the NOD mouse. Dr. Davis, in collaboration with Dr. Chien has developed techniques for polymerase chain reaction analysis of single T cells isolated from 14-18 day old NOD mice. This has lead to determination of sequences utilized by the T cell receptors in these T cells, and ultimately will lead to the production of transgenic mice expressing insulitis-inducing T cell receptors expressed early in the course of type I IDDM. This project requires sophisticated cell sorting to gate out all of the extraneous cells in islet preparations to isolate the relatively small number of T cells present in these islets at the very beginning of the diabetogenic process. Dr. Crabtree, in collaboration with Dr. McDevit will produce NOD mice expressing mutant cyclophilins which will permit blockade of T cell receptor signaling at various time points in the development of diabetes in NOD mice. Fluorescent Activated Cell Sorting (FACS) analysis will characterize the resultant T cell populations in these immunosupressed and islet cell antigen tolerant animals. Similarly, the experiments proposed by Dr. Weissman and Shizuru will require sophisticated cell sorting to isolate hematopoietic stem cell for stem cell transplantation into young NOD mice, utilizing stem cells and whole bone marrow from H2-matched as well as syngeneic donors. The development of the T cell receptor repertoire in this mice will be monitored using the staining reagents developed by Dr. Chien, the single cell analysis of T cell receptor sequences developed by Drs. Chien and Davis, and the methods for characterizing the T cell receptors repertoire utilized by Dr. Crabtree.
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|Shizuru, J A; Weissman, I L; Kernoff, R et al. (2000) Purified hematopoietic stem cell grafts induce tolerance to alloantigens and can mediate positive and negative T cell selection. Proc Natl Acad Sci U S A 97:9555-60|
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