Characterization Of Gene Expression In Mouse Resting And Activated Mast Cells And Molecular Cloning Of A Novel Mast Cell Serine Protease-10

Characterization of gene expression in mouse resting and activated mast cells and Molecular cloning of a novel mast cell serine protease-10ObjectiveMast cells are important effector cells in IgE-associated allergic reactions. During the process of allergic reaction, the expression level of some regulated genes will be changed and mast cells may release of preformed granule mediators, multiple cytokines, chemokines and other immunomodulatory molecules. All these changes are related to activation of mast cells. Early studies on mast cells were largely concerned with examining functional or biochemical phenomena, more recently some genes coding for molecules thought to be involved in the activation of mast cells have been identified and in a few instances manipulated to determine the role of the proteins they code for. The aim of the present investigation was to define comprehensively the profiles of expressed genes in mouse resting and activated bone marrow derived mast cells (BMMC) in order to discover evidences for studying new mechanism and new therapeutic method of allergic reaction.Method1.There is a method termed SST-REX (signal sequence trap by retrovirus-mediated expression screening). Its retrovirus vector pME-SST has the ability to direct a constitutively active mutant thrombopoietin receptor MPL (MPL~m) to the membrane of cells, thereby allowing interleukin-3 (IL-3)-independent growth of otherwise IL-3-dependent pro-B cell line Ba/F3. We had exploited this property of MPL~m to sreen mouse resting and activated BMMC cDNA libaries with the vector pMX-SST.2. After the genomic DNA of Ba/ F3 cells with IL-3-independent growth was isolated, the resting and activated BMMC cDNAs, which were inserted into Ba/F3 cells, were recoveried by polymerase chain reaction (PCR).3.The PCR fragments were sequenced with Sanger method, then, we analyze the PCR fragments' sequences in gene bank.4. To analyze the expression levels of unknown genes, cDNA from mouse resting and activated mast cells were hybrized with the unknown genes fragments.5. The full-length of unknown genes were cloned with RecA mediated clone capture.Results1. It is important that we identified 54 known genes, which had confirmed to...