The Drosophila Gene Tagging Project: Transgenes with Swappable Protein Tags

描述(由申请人提供)： 在操纵果蝇基因组方面的最新进展正在为这一关键模式生物的高通量分子遗传学创造新的可能性。其中最重要的进展是通过重组工程修改基因组克隆的P[Acman]方法，然后将得到的克隆特定位置整合到苍蝇基因组中。 P[Acman]最有用的应用之一是将标签结合到基因的开放阅读框架中来表达融合蛋白。蛋白质标签有许多应用，包括可视化表达模式，以及免疫沉淀蛋白质复合体、RNA-蛋白质复合体和染色质。新技术在果蝇研究中的广泛和有效使用依赖于DNA克隆库和转基因果蝇品系等资源，这些资源极大地提高了生产率。我们建议开发一种新型的多功能和可互换的蛋白质标签盒，将标签整合到21kb的BAC克隆中，创建400个研究最多的果蝇基因的基因组融合构建体，产生表达标签蛋白的转基因苍蝇系，并将标记的BAC和苍蝇系分发给研究社区。在以前的工作中，我们建立了使用P[Acman]进行基因标记的可行性。这项提议将为扩大这种方法的规模奠定基础，以实现对基因、相互作用和途径的高通量遗传操作。新的蛋白质标签的一个关键特征是结合了重组酶介导的盒式交换(RMCE)，用于将转基因果蝇品系内的标签与任何其他设计的DNA盒交换，以创建定制的标记等位基因，从而极大地扩大了标记菌株集合的实用性。总之，新的可交换蛋白质标签和高通量重组工程和转基因管道将证明为大多数果蝇基因产生基因组规模的蛋白质标签等位基因集合的可行性，证明含有RMCE的单一果蝇品系集合几乎可以支持标记蛋白质的所有应用，并将果蝇的功能基因组学转移到任何其他后生动物模式生物无法比拟的更高平台上。最后，由于P[Acman]和标签的成分可以在许多其他物种中发挥作用，我们的方法是包括鼠和人类细胞培养在内的其他物种的模型。 公共卫生相关性： 美国国立卫生研究院人类基因组计划制作了人类DNA序列和用于医学研究的关键动物中编码的蛋白质的完整列表。这些蛋白质是生命的基石，但超过一半的蛋白质在细胞和组织中的作用鲜为人知。为了加快蛋白质功能的研究，我们将对一种关键的模式动物--果蝇黑腹果蝇--的基因组进行改造，以生产这些蛋白质的标记版本。这些菌株将允许研究人员探索它们在哪里被激活，以及它们与哪些其他蛋白质相互作用。这类基础研究是促进未来人类健康的基础。

DESCRIPTION (provided by applicant): Recent advances in manipulation of the Drosophila melanogaster genome are creating new possibilities for high-throughput molecular genetics in this key model organism. Among the most significant advance is the P[acman] methodology for modification of genomic clones via recombineering, followed by site- specific integration of the resulting clones into the fly genome. One of the most useful applications of P[acman] is to incorporate tags within open reading frames of genes to express fusion proteins. Protein tags have many applications, including visualizing expression patterns, and immunoprecipitation of protein complexes, RNA- protein complexes and chromatin. The widespread and efficient use of new technologies in Drosophila research has depended on resources such as libraries of DNA clones and transgenic fly lines, which have dramatically increased productivity. We propose to develop a novel multifunctional and exchangeable protein tagging cassette, incorporate the tag into 21-kb BAC clones to create genomic fusion constructs for the 400 most studied Drosophila genes, generate transgenic fly lines expressing tagged proteins, and distribute the tagged BACs and fly lines to the research community. In previous work, we established the feasibility of gene tagging using P[acman]. This proposal will establish the basis for scaling up this approach for high-throughput genetic manipulation of genes, interactions, and pathways. A key feature of the new protein tag is the incorporation of Recombinase Mediated Cassette Exchange (RMCE) for swapping the tag within the transgenic fly line with any other designer DNA cassette to create custom tagged alleles, thus dramatically expanding the utility of the collection of tagged strains. In sum, the new exchangeable protein tag and the high-throughput recombineering and transgenesis pipeline will demonstrate the feasibility of generating a genome-scale collection of protein tag alleles for most Drosophila genes, demonstrate that a single collection of fly strains that incorporates RMCE can support virtually all applications of tagged proteins, and move the functional genomics of Drosophila to a higher plateau unmatched by any other metazoan model organism. Finally, as the components of P[acman] and the tag can function in many other species, our approach is a model for other species including the mouse and human cell culture. PUBLIC HEALTH RELEVANCE: The NIH Human Genome Project produced complete lists of the proteins encoded in the DNA sequences of humans and also in key animals used in medical research. These proteins are the building blocks of life, but more than half have poorly understood roles in cells and tissues. To accelerate research into the functions of proteins, we will engineer the genome of a key model animal - the fruit fly Drosophila melanogaster - to produce tagged versions of these proteins. These strains will allow investigators to explore where they are turned on and with what other proteins they interact. Basic studies such as these are fundamental to advancing human health in the future.