RESOLVING COMPLEX SYSTEMIC ENDOGENOUS EXPRESSION PATTERNS INTO SUBCELLULAR HIGH-RESOLUTION LOCALIZATION

项目摘要 表型、基因组和蛋白质组学分析的结合已经很容易地推进了确定 在基因上易处理的生物体如果蝇中的基因功能。然而，基因 对复杂的生理和发育过程的分析可能会被以下几个因素所掩盖， 蛋白质的生化功能丧失和表型变得明显之间的延迟。 蛋白质内源性表达的详细成像对于提供其内源性表达的完整描述是必不可少的。 在发育、生理甚至病理事件的动态过程中发挥作用。一方面， 检查体内表达的显微镜方法通常提供表达的静态视图。另 另一方面，与荧光标记融合的基因的异位表达可能具有来自错误表达的假象。 科学家们需要的是一种简单的方法来产生一种内源性标记蛋白质，理想情况下， 通过简单的遗传操作将表达分解到单个细胞。本提案提出了这样一种方法 并使用果蝇进行验证，其中标记的基因可以放置在不同的突变背景中， 检测蛋白质表达的动态差异。作者使用具有转座因子的果蝇 插入到确定的遗传位点上。插入被用作靶位点以稳定地插入人工外显子，所述人工外显子编码 几种荧光蛋白中的一种，通过RNA剪接很容易产生标记的基因融合。一个 替代标签使基因的RNA产物而不是其蛋白质产物可追踪。第三个标签有助于 蛋白质在其天然表达条件下的生物化学纯化。此外，表达 个体果蝇中标记的融合蛋白随后从整个生物体缩小到簇 甚至是单个细胞。这个精确的表达式是通过实验控制两个 重组酶的组织特异性和时间调节，所述重组酶靶向位于人工外显子编码 标签重组酶控制调节可观察基因表达的开关。这个开关可以是 用FLP和Cre重组酶有条件地打开“ON”和“OFF”。最后，将产生试剂， 将通过简单的交叉促进可转换试剂的交换，使该方法对任何苍蝇都适用。 遗传学家总之，该提议旨在开发超过100个内源性标记基因的集合， 18个表达调控重组酶的转基因原种和24个允许靶向重组酶的转基因原种。 果蝇中任何带有所提议的标签组的基因。这些试剂将免费提供给 研究社区。虽然这种方法是为苍蝇开发的，但它可以很容易地扩展到其他生物。

Project Summary The combination of phenotypic, genomic and proteomic analyses have readily advanced the determination of gene function in genetically tractable organisms such as Drosophila melanogaster. However, genetic analysis of complex physiological and developmental processes can be obscured by several factors including the delay between when a protein's biochemical function is lost and when a phenotype becomes apparent. Detailed imaging of a protein's endogenous expression is essential to provide a complete description of its function during the dynamics of developmental, physiological and even pathological events. On one hand, microscopic methods to examine expression in vivo generally provide a static view of expression. On the other hand, ectopic expression of a gene fused with a fluorescent marker can have artifacts from mis-expression. What scientists need is an easy method to generate an endogenously tagged protein, ideally one that can resolve expression to individual cells by simple genetic manipulation. This proposal develops such a method and validates it using Drosophila where the tagged gene can be placed in different mutant backgrounds to examine dynamic differences in protein expression. The authors use flies that have transposable elements inserted at defined genetic loci. The insertions are used as target sites to stably insert artificial exons encoding one of several fluorescent proteins that readily create a tagged gene fusion through RNA splicing. An alternative tag makes the gene's RNA product traceable rather than its protein product. A third tag facilitates the biochemical purification of a protein under its native expression conditions. Additionally, the expression of the tagged fusion protein in individual flies is subsequently narrowed down from the whole organism to clusters of cells or even individual cells. This refined expression is derived by the experimental control of both the tissue-specific and temporal regulation of recombinases that target sites flanking the artificial exons encoding the tags. The recombinases control a switch that regulates observable gene expression. This switch can be conditionally turned “ON” and “OFF” with FLP and Cre recombinases. Finally, reagents will be generated that will facilitate exchange of switchable reagents through simple crosses, making the method accessible to any fly geneticist. In conclusion, this proposal aims to develop a collection of over 100 endogenously tagged genes, 18 transgenic stocks expressing regulated recombinases and 24 transgenic stocks that permit the targeting of any gene in the fly with the proposed panel of tags. These reagents will be made freely available to the research community. While developed for flies, this method can readily be extended to other organisms.