| Research Abstract |
Large scale monitoring of gene expression is a powerful approach to clarify the cellular events. DNA microarray technologies permit us to recognize genome-wide expression pro filing, and bring a profound impact to biological research, such as pharmacology. This technology can also be applied to drug discovery and molecular classification of diseases. We introduced microarray to discover novel function of genes involved in disease states. In analyzing tissues of an animal disease model, we first attempted to use microarrays with cDNAs randomly selected from Unigene clones (Research Genetics), and found that few clones successfully hybridized presumably because of the tissue-specific gene expression. To resolve this problem, we aimed to fabricate microarrays with cDNA library of the tissue to be analyzed ; however, ordinary cDNA libraries contain a high frequency of undesirable clones because of redundancy of mRNA species in the cell. To get rid of the redundancy from ordinary cDNA libra
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ries ("normalization"), we chose two approaches of "subtractive library" and "hit-picking". "Subtractive library" approach is a conventional method, while "hit-picking" approach is a novel one we developed by combining macroarray and robotic systems ; we named the latter method "hit-picking" since we collect desirable clones after selection by filter hybridization. By using DNA chip with normalized cDNA library, we analyzed IgA nephropathy model animal. The molecular mechanism of immunoglobulin A nephropathy (IgAN), the most common primary renal glomerular disease worldwide, is unknown. HIGA (high serum IgA) mouse is a valid model of IgAN showing almost all of the pathological features, including mesangial cell proliferation. Here we elucidate a pattern of gene expression asosociated with IgAN by analyzing the diseased kidneys on cDNA microarrays. In particular, we showed an enhanced expression of several genes regulating the cell cycle and proliferation, including growth factors and their receptors, as well as endothelial differentiation gene-5 (EDG5), a receptor for sphingosine 1-phosphate (SPP). One of growth factors, platelet-derived growth factor (PDGF) induces a marked upregulation of EDG5 in proliferative mesangial cells, and promotes cell proliferation synergistically with SPP. The genomic approach allows us to identify families of genes involved in a process, and can indicate that an enhanced PDGF-EDG5 signaling plays an important role in the progression of IgAN. Less
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