A New Tool Kit for Whole-Genome Mapping and Complementation in Drosophila

描述（由申请人提供）：本提案的最终目标是生成、维护和分发一个新的复制品库，该复制品库几乎涵盖了黑腹果蝇基因组的整个常染色体部分。由于这一收集将代表一套强大的新工具，将有利于几乎所有的果蝇遗传学家，很可能会有一个高需求的这一资源。我们对人类发育的理解中许多最重要的进展都来自于使用果蝇作为动物模型系统的研究。由于果蝇和哺乳动物在控制生物过程的潜在分子机制方面存在许多相似之处，因此从果蝇研究中获得的知识可以直接应用或容易地适用于理解人类生物学。 我们建议生成一个新的重复集合，连同现有的X和第4染色体集合，将覆盖整个果蝇基因组的99.7%。这种新的复制集支持跨不同领域的广泛应用。首先，它将允许快速定位和鉴定正常发育和功能所需的新基因。与目前使用分子定义的果蝇常染色体缺失试剂盒（MDADK）的作图方法相比，这种重复收集将提供平均高10倍的分辨率。由于该复制试剂盒基于产生携带基因组构建体的转基因果蝇，因此可以对相同构建体进行进一步操作以用于下游功能分析，包括蛋白质的标记和结构-功能研究。此外，这些股票可用于遗传拯救实验，这对于分配基因功能以及测试和验证世界各地股票中心持有的大量突变体至关重要。我们估计总共需要3，315个转基因种群来完成这一收集，我们将该项目分为两个阶段：第一阶段已经获得资助并接近完成：拟议的400个BAC中的350个已经注射，263个转基因分离。在第二阶段，我们将完成收集。有三个具体目标：目标1。选择并验证覆盖2号和3号染色体的BAC平铺阵列。目标2.生成并验证完整的常染色体复制试剂盒。目标3。创建和维护一个在线可搜索的BAC和转基因数据库。 第一阶段的成功填补了目前MDADK中74%的空白，也为扩大我们的努力创造了完整的集合。利用新技术的好处，这个集合将是果蝇研究工具包的另一个关键补充。作为世界上大多数果蝇遗传学家高度期望的资源，该集合将为整个果蝇研究社区提供巨大的实用性，其应用范围包括遗传精细作图，基因组拯救，体细胞克隆分析和基因组标记。

DESCRIPTION (provided by applicant): The ultimate goal of this proposal is to generate, maintain, and distribute a new collection of duplication stocks that covers nearly the entire euchromatic autosomal portion of the Drosophila melanogaster genome. Since this collection will represent a powerful new set of tools that will benefit virtually all Drosophila geneticists, t is likely that there will be a high demand for this resource. Many of the most important advances in our understanding of human development have come from studies using the fruit fly Drosophila melanogaster as an animal model system. Since many parallels exist between Drosophila and mammals in terms of the underlying molecular mechanisms controlling biological processes, knowledge gained from research in Drosophila can be either directly applied or readily adapted to understanding human biology. We propose to generate a new duplication collection that, together with existing collections for the X and 4th chromosomes, will cover 99.7% of the entire Drosophila genome. This new duplication set supports a broad range of applications across different fields. First, it will allow rapid mapping and identification of new genes required for normal development and function. Compared to the current mapping approach that uses the molecularly defined Drosophila autosomal deletion kit (MDADK), this duplication collection will offer an average of 10-fold higher resolution. As this duplication kit is based on generating transgenic flies carrying genomic constructs, further manipulation of the same constructs can be performed for downstream functional analysis, including tagging of proteins and structure-function studies. Moreover, these stocks can be used for genetic rescue experiments that are essential for assigning gene function as well as testing and validating the huge collection of mutants held in stocks centers around the world. We estimate that a total of 3,315 transgenic stocks will be needed to complete this collection and we split this project into two phases: Phase I has been funded and is nearly complete: more than 350 of the proposed 400 BACs have been injected and 263 transgenics isolated. Here in Phase II, we will complete the collection. There are three Specific Aims: Aim 1. Select and validate a tiled array of BACs covering chromosomes 2 and 3. Aim 2. Generate and validate a complete autosomal duplication kit. Aim 3. Create and maintain an online searchable database for BACs and transgenics. Success in Phase I has filled 74% of the bases in gaps in the current MDADK, and has also set the stage for expanding our effort to generate the complete collection. Harnessing the benefits of new technologies, this collection will be another key addition to the Drosophila research tool kit. As a highly desired resource by most Drosophila geneticists across the world, this collection will offer great utility or the entire Drosophila research community, with applications ranging from genetic fine mapping, genomic rescue, somatic clonal analysis, and genomic tagging.