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

Wnt-无翅（Wg）途径调节后生动物发育的许多方面。异常的Wnt信号传导与人类疾病如肝癌、结肠直肠癌、乳腺癌和皮肤癌有关。遗传学和生物化学方法已经确定了许多调节果蝇和其他模式生物中Wnt-Wg途径的基因。然而，许多组分可能仍未鉴定。本研究中使用的RNAi筛选试验基于Wnt报告基因TOP-Flash（TCF最佳启动子），其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。在果蝇S2 R+细胞中进行筛选，其中Wg途径是活跃的，因此S2 R+细胞可能含有响应Wg所需的大部分组分。该测定涉及TOP-Flash报道基因的转染，连同作为转染效率对照的海肾荧光素酶载体和编码wg（pMK 33-wg）的表达载体一起沿着转染，以刺激细胞的增殖。 关于我们 通路通过测量归一化的荧光素酶表达或相对荧光素酶活性单位来定量Wg信号传导途径的活性，相对荧光素酶活性单位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的比率。RNAi介导的敲除正调控抑制Wg增强的报告活性，而RNAi敲除负调控在无刺激的情况下异位激活报告，或在Wg诱导时进一步协同激活报告。因此，我们可以使用该报告基因来鉴定正性和负性调节剂。我们在S2 R+细胞中进行了全基因组RNAi筛选，以筛选Wnt途径的调节因子。我们确定了新的180个（148个阳性和32个阴性）潜在的监管机构。总的来说，在培养细胞中进行高通量RNAi筛选，然后在模型生物中进行功能分析，证明是鉴定与发育和疾病有关的信号通路调节剂的快速方法。少

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