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

使用全基因组 RNAi 对 JAK/STAT 信号传导进行功能分析

• 批准号: 5446283
• 负责人: Professor Dr. Michael Boutros
• 金额: --
• 依托单位: Abteilung Signalwege und funktionelle Genomik (B110)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2005
• 资助国家: 德国
• 起止时间: 2004-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5446283?language=en
• 关键词: Functional; analysis; JAK; STAT; signalling

The JAK/STAT signal transduction pathway is involved in development, immunity anddisease and has been conserved throughout evolution. While components of both theDrosophila and mammalian JAK/STAT pathway have been characterized by biochemical andgenetic means, a number of significant gaps in our understanding of the pathway remain. Herewe propose experiments to comprehensively identify the genes involved in poorly understoodaspects of pathway signalling using Drosophila as a model system. First, using an availableRNAi libary targeting every gene in the fly genome, we will sreen for JAK/STATcomponents in Drosophila macrophage-like cells. To this end, we have established a reportergene assays that accurately reflects JAK/STAT pathway activity. We will then re-screen thepotentially interacting genes identified during the initial screen to determine those directlyimplicated in our specific fields of research. These interacting genes will be evaluated andprioritized before analysis using genetic and biochemical experiments. Martin Zeidler willfurther identify genes involved in upstream processes of ligand modification, secretion,receptor binding and internalisation. This will employ a GFP-tagged ligand which can bevisualised both in cultured cells in vivo. Mutations in the genes affecting these processes willbe 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 immuneresponse, although its in vivo function remains poorly understood. Loss-of-functionphenotypes of novel components will be studied in vitro.

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一直是保守的。虽然果蝇和哺乳动物JAK/STAT通路的组成部分已经通过生物化学和遗传学手段进行了表征，但我们对该通路的理解仍然存在许多重大差距。在此，我们提出了实验，以全面确定基因参与的不太了解方面的途径信号使用果蝇作为一个模型系统。首先，利用一个可利用的靶向果蝇基因组中每一个基因的RNAi库，我们将在果蝇类巨噬细胞中筛选JAK/STAT组分。为此，我们建立了一种准确反映JAK/STAT通路活性的转基因检测方法。然后，我们将重新筛选在初步筛选中发现的潜在相互作用基因，以确定那些直接涉及我们特定研究领域的基因。这些相互作用的基因将在使用遗传和生化实验进行分析之前进行评估和优先排序。Martin Zeidler将进一步鉴定参与配体修饰、分泌、受体结合和内化的上游过程的基因。这将采用GFP标记的配体，其可以在体内培养的细胞中可视化。影响这些过程的基因突变将被分析，以确定它们在体内与JAK/STAT依赖表型的相互作用。Michael Boutros将分析新发现的基因在先天免疫中的作用。JAK/STAT信号转导已被我们认为是果蝇先天免疫应答的一部分，尽管其在体内的功能仍然知之甚少。将在体外研究新组分的功能丧失表型。