Mechanisn of lymphocyte differentiation, molecular mechanisms of gene rearrangement and clonal deletion by antigenes

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This year we have extended our previou studies using signal sequence trap method which allowed us to isolate a number of novel growth factors and their receptors. We are currently producing protein products encoded by isolated genes. These products will be added to the in vitro coculture system which will be described bellow. We have developed the new coculture system which allows to differentiate ES cells into all lineages of hematopoietic cells including lymphocytes. ES cells normally protected from differentiation by LIF was induced to differentiate by coculturing with a stroma cell line OP9 which cannot produce M-CSF because M-CSF inhibits differentiation into all the other hematopoietic lineages. Under this condition ES cells differentiate into red blood cells, granulocytes, megakaryocytes and lymphocytes. We have shown that lymphocytes actually express surface IgM.We have also shown several genes were induced upon programd cell death. One of such genes named MA3 was isolated and the sequence data indicate that MA3 is a novel gene with novel function. We have also isolated several known genes such as genes for heat shock protein. Another novel gene isolated in our laboratory about five years ago, colled RBP-Jk was originally assumed to be involved in immunoglobulin gene recombination because of the presence of the integrase-related motif. Knock out experiments and transgenic fry experiments indicate that the RBP-Jk gene is involved in signal transduction from the Notch receptor which regulates peripheral nervous development in Drosophila and probably lymphocyte proliferation in mammals. We have shown that RBP-Jk is involved in lateral inhibition of Drosophia and interacts with EBNA2 encoded by EB virus.