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

今年，我们使用信号序列陷阱方法扩展了我们以前的研究，该方法使我们能够分离出许多新的生长因子及其受体。我们目前正在生产由分离基因编码的蛋白质产品。这些产品将被添加到体外共培养系统，这将在下面描述。我们已经开发了新的共培养系统，可以将胚胎干细胞分化为包括淋巴细胞在内的所有造血细胞。通过与基质细胞系OP9共培养诱导胚胎干细胞分化，而基质细胞系OP9不能产生M-CSF，因为M-CSF抑制向所有其他造血谱系的分化。在这种条件下，胚胎干细胞分化为红细胞、粒细胞、巨核细胞和淋巴细胞。我们已经证明淋巴细胞实际上表达表面IgM。我们还表明，在程序性细胞死亡时，有几个基因被诱导。其中一个名为MA3的基因被分离出来，序列数据表明MA3是一个具有新功能的新基因。我们还分离出了一些已知的基因，如热休克蛋白基因。另一个新基因colcoldrbp - jk是我们实验室在大约5年前分离出来的，由于整合酶相关基序的存在，最初认为它参与了免疫球蛋白基因重组。敲除实验和转基因苗实验表明，RBP-Jk基因参与Notch受体的信号转导，Notch受体在果蝇中调节周围神经发育，也可能在哺乳动物中调节淋巴细胞增殖。我们已经证明RBP-Jk参与果蝇的侧抑制，并与EB病毒编码的EBNA2相互作用。

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.