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RNAi-based genome-wide search for genes constituting Wnt signaling pathway by using Drosophila tissue culture cells

利用果蝇组织培养细胞进行基于RNAi的全基因组搜索构成Wnt信号通路的基因

基本信息

批准号：
17570111
负责人：
YANAGAWA Shin-ichi
金额：
$ 2.3万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570111/
关键词：
RNAi Wnt Wingless Signal Transduction Genome Cell Culture Reporter Assay

项目摘要

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 … More 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

Wnt-Wingless (Wg) 通路调节后生动物发育的许多方面。异常的 Wnt 信号传导与肝癌、结直肠癌、乳腺癌和皮肤癌等人类疾病有关。遗传和生化方法已经鉴定出果蝇和其他模式生物中调节 Wnt-Wg 通路的许多基因。然而，许多成分可能仍未确定。本研究中使用的 RNAi 筛选测定基于 Wnt 报告基因 TOP-Flash（TCF 最佳启动子），它由驱动编码萤火虫荧光素酶基因的 cDNA 表达的多聚化 TCF 结合位点组成。该筛选是在果蝇 S2R+ 细胞中进行的，其中 Wg 途径处于活跃状态，因此 S2R+ 细胞可能含有响应 Wg 所需的大部分成分。该测定涉及 TOP-Flash 报告基因的转染，以及作为转染效率对照的海肾荧光素酶载体，以及编码 wg (pMK33-wg) 的表达载体以刺激通路。 Wg信号通路的活性通过测量标准化荧光素酶表达或相对荧光素酶活性单位来量化，其等于萤火虫荧光素酶的绝对活性与海肾荧光素酶的绝对活性之比。 RNAi 介导的正调节因子敲低抑制了 Wg 增强的报告基因活性，而 RNAi 敲低负调节因子在没有刺激的情况下异位激活报告基因，或者在 Wg 诱导时进一步协同激活报告基因。因此，我们可以使用该报告基因来识别正调节剂和负调节剂。我们在 S2R+ 细胞中进行了全基因组 RNAi 筛选，以筛选 Wnt 通路的调节因子。我们确定了 180 个新的潜在调节因子（148 个正调节因子和 32 个负调节因子）。总而言之，在培养细胞中进行高通量 RNAi 筛选，然后在模式生物中进行功能分析，被证明是识别与发育和疾病有关的信号通路调节因子的快速方法。较少的