Genome-wide quantitative genetic analysis of growth and starvation survival

描述（由申请人提供）： 阻止生长和在饥饿中生存的能力是与癌症、糖尿病和衰老相关的基本特征。后生动物生长和繁殖所需的基因已经被全面鉴定，但相对较少的基因饥饿生存所必需的是已知的，只有少数增加生存已知。目前尚不清楚哪些途径是中心的，哪些是缺失的，以及这些途径如何整合以产生对养分可用性的协调反应。本项目的长期目标是确定作为数量性状的耐饥饿性的遗传结构。这包括全面鉴定影响饥饿生存的基因以及它们的成对上位相互作用。然而，现有的后生动物遗传分析技术是相对费力的，而不是定量的，上位性分析的方法通常集中在个体的遗传相互作用。我们正在开发一种大规模平行的方法来测量基因组中每个基因对C.优雅基本原理是使用转座子对大群体进行插入诱变，选择感兴趣的表型（饥饿存活），并对转座子侧翼进行深度测序以测量等位基因频率。C.线虫是这个项目的理想选择，因为线虫适应于在盛宴和饥荒的循环中生存，有丰富的遗传工具包可以用于这种创新方法，基因组紧凑，可以培养非常大的种群，并且已经全面鉴定了必需基因，便于验证。我们使用的Mos 1转座子是理想的，因为它在不存在转座酶的情况下是稳定的，其突变率自然低且可优化，并且经典的正向遗传方案已经到位。使用基于模型的模拟的初步结果表明，我们将能够在一次试验中多次突变几乎每个基因，并有能力检测群体之间等位基因频率的微小差异。该提案的目标是开发和验证这种方法，并全面鉴定增加或减少饥饿抗性的基因。我们假设许多在传统筛选中没有明显表型的基因对饥饿生存至关重要，但相对较少的基因会增加生存。我们将通过以下三个具体目标来实现我们的目标：1）在C中开发转座子介导的测序遗传学（Gen-Seq）。elegans，2）通过全面鉴定必需基因进行的基因测序和3）全面鉴定修饰饥饿抗性的非必需基因。我们提出了初步的建模结果和协议设计的可行性支持。该建议是创新的，利用下一代测序和模型系统遗传学的力量来开发一种方法，这将大大加快遗传分析。结果将是显着的，因为饥饿生存的遗传基础还没有在后生动物中确定，虽然它是一个基本特征与巨大的疾病相关性。未来的工作将利用Gen-Seq进行基因组范围内的遗传相互作用的鉴定，这些相互作用是饥饿抗性的基础。

DESCRIPTION (provided by applicant): The ability to arrest growth and survive starvation is a fundamental trait relevant to cancer, diabetes and aging. Metazoan genes required for growth and fertility have been comprehensively identified, but relatively few genes essential to starvation survival are known, and only a handful that increase survival are known. It is un- clear which pathways are central, which are missing, and how these pathways are integrated to produce coordinated responses to nutrient availability. The long-term goal of this project is to determine the genetic architecture f starvation resistance as a quantitative trait. This includes comprehensive identification of genes that influence starvation survival as well as their pair-wise epistatic interactions. However, existing technologies for metazoan genetic analysis are relatively laborious and not quantitative, and approaches to epistasis analysis generally focus on individual genetic interactions. We are developing a massively parallel method to measure the functional contribution of each gene in the genome to a phenotype of interest in C. elegans. The rationale is to use a transposon for insertional mutagenesis of a large population, select for the phenotype of interest (starvation survival), and deep sequence transposon flanks to measure allele frequencies. C. elegans is ideal for this project since nematodes are adapted to survive cycles of feast and famine, there is a rich genetic toolkit that can be leveraged for this innovative approach, the genome is compact, extremely large populations can be cultured, and essential genes have been comprehensively identified facilitating validation. The Mos1 transposon we are using is ideal in that it is stable n the absence of transposase, its mutation rate is naturally low and optimizable and classic forward genetic protocols are in place. Preliminary results using model-based simulation suggest we will be able to mutate almost every gene multiple times in a single trial and have the power to detect small differences in allele frequency between populations. The objectives of this proposal are to develop and validate this approach and to comprehensively identify genes that increase or decrease starvation resistance. We hypothesize that many genes with no apparent phenotype in traditional screens will be essential to survive starvation but that relatively few genes will increase survival. We will accomplish our objectives with the following three specific aims: 1) Develop transposon-mediated genetics by sequencing (Gen-Seq) in C. elegans, 2) Validate Gen-Seq by comprehensively identifying essential genes and 3) Comprehensively identify non-essential genes that modify starvation resistance. We present preliminary modeling results and protocol designs in support of feasibility. This proposal is innovative for leveraging the power of next-generation sequencing and model system genetics to develop a methodology that will greatly expedite genetic analysis. The results will be significant since the genetic basi of starvation survival has not been determined in a metazoan, though it is a fundamental trait with tremendous disease relevance. Future work will take advantage of Gen- Seq for genome-wide identification of genetic interactions underlying starvation resistance.