Functional analysis of JAK/STAT signalling using genome-wide RNAi

JAK/STAT信号转导通路参与发育、免疫和疾病，并且在整个进化过程中都是保守的。虽然果蝇和哺乳动物JAK/STAT途径的组成成分已经通过生化和遗传手段进行了表征，但我们对该途径的理解仍然存在一些重大差距。在这里，我们建议以果蝇为模型系统，进行实验，以全面识别涉及鲜为人知的途径信号方面的基因。首先，利用现有的针对果蝇基因组中每一个基因的RNAi文库，我们将在果蝇巨噬细胞样细胞中筛选JAK/STAT组分。为此，我们建立了一种准确反映JAK/STAT途径活性的报告基因分析方法。然后，我们将重新筛选在初始筛选过程中确定的潜在相互作用的基因，以确定那些与我们的特定研究领域直接相关的基因。在使用遗传和生化实验进行分析之前，将对这些相互作用的基因进行评估和优先排序。Martin Zeidler将进一步识别参与配体修饰、分泌、受体结合和内化等上游过程的基因。这将使用GFP标记的配体，在体内培养的细胞中都可以看到。影响这些过程的基因突变将被分析，以确定它们与体内JAK/STAT依赖表型的相互作用。迈克尔·布特罗斯将分析新发现的基因在先天免疫中的作用。JAK/STAT信号被我们认为是果蝇先天免疫反应的一部分，尽管它在体内的功能仍然知之甚少。新成分的功能丧失表型将在体外进行研究。

The JAK/STAT signal transduction pathway is involved in development, immunity and disease and has been conserved throughout evolution. While components of both the Drosophila and mammalian JAK/STAT pathway have been characterized by biochemical and genetic means, a number of significant gaps in our understanding of the pathway remain. Here we propose experiments to comprehensively identify the genes involved in poorly understood aspects of pathway signalling using Drosophila as a model system. First, using an available RNAi libary targeting every gene in the fly genome, we will sreen for JAK/STAT components in Drosophila macrophage-like cells. To this end, we have established a reporter gene assays that accurately reflects JAK/STAT pathway activity. We will then re-screen the potentially interacting genes identified during the initial screen to determine those directly implicated in our specific fields of research. These interacting genes will be evaluated and prioritized before analysis using genetic and biochemical experiments. Martin Zeidler will further identify genes involved in upstream processes of ligand modification, secretion, receptor binding and internalisation. This will employ a GFP-tagged ligand which can be visualised both in cultured cells in vivo. Mutations in the genes affecting these processes will be analysed to determine their interactions with JAK/STAT dependent phenotypes in vivo. Michael Boutros will analyze newly identified genes for their role during innate immunity. JAK/STAT signalling have been implicated by us as part of the Drosophila innate immune response, although its in vivo function remains poorly understood. Loss-of-function phenotypes of novel components will be studied in vitro.