The finding that more than 152 tissue-restricted antigens are expressed by thymic medullary epithelial cells is redefining the importance of thymic central tolerance induction in the prevention of autoimmune diseases. One of the tissue-restricted antigens in the thymus is proinsulin, and in both mice and humans, reduced thymic proinsulin levels have been shown to predispose to Type 1 diabetes. Using transgenic mice expressing a functional beta-Galactosidase gene under the regulation of the Ins2 promoter we have determined that between 1-3% of all medullary thymic epithelial cells express proinsulin and that these cells are frequently part of the Hassall's Corpuscles like structures in mice. Using a cross between the beta-Galactosidase expressing mice and Immortomice (expressing SV40 large T Antigen under the regulation of the MHC I promoter), we have isolated and cultured two proinsulin and two non-proinsulin producing medullary epithelial cell lines. Microarray analysis and RT-PCR analysis of the cell lines revealed the over-expression of approximately 50 genes (>4 fold or more) in the proinsulin producing lineage, versus the non proinsulin producing lineage, and approximately half the over-expressed genes can be considered tissue-restricted antigens. We do not find any evidence for chromosomal clustering of the over-expressed genes nor do we report the expression of any other pancreatic n-cell antigens or specific pancreatic proinsulin regulatory proteins (Pdx-1, Glut-2 or GCK) within the proinsulin producing cell lines but we do detect their expression in whole thymus. Our results suggest that chromosomal clustering is not a phenomenon associated with thymic tissue-restricted antigen expression and that the mechanisms allowing for thymic tissue-restricted antigen expression are not related to the expression mechanisms of such antigens in peripheral tissues.

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