| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
The Wnt-Wingless (Wg) pathway regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease such hepatic, colorectal, breast, and skin cancers. Genetic and biochemical approaches have identified many of the genes that regulate the Wnt-Wg pathway in Drosophila and other model organisms. However, many components may remain unidentified The assay for the RNAi screen used in this study was based on the Wnt reporter TOP-Flash (TCF optimal promoter), which consists of multimerized TCF-binding sites driving the expression of a cDNA encoding the firefly luciferase gene. The screen was performed in Drosophila S2R+ cells, in which the Wg pathway is active and thus S2R+ cells are likely to contain the majority of the components required to respond to Wg. The assay involved transfection of the TOP-Flash reporter, along with a Renilla luciferase vector as a control for transfection efficiency, and an expression vector encoding wg (pMK33-wg) to stimulate the
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pathway. The activity of the Wg signaling pathway was quantified by measurement of normalized luciferase expression or relative luciferase activity units, which equated to the ratio of the absolute activity of firefly luciferase to that of renilla luciferase. RNAi-mediated knockdown of positive regulators suppressed Wg-enhanced reporter activity, whereas RNAi-knockdown of negative regulators ectopically activated the reporter in the absence of stimulus or further synergistically activated the reporter when induced by Wg. Thus, we could use this reporter to identify both positive and negative modulators. We performed a genome-wide RNAi screen in S2R+ cells to screen for regulators of the Wnt pathway. We identified novel 180 (148 positive and 32 negative) potential regulators. Over all, high-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease. Less
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