Genome-wide quantitative genetic analysis of growth and starvation survival

生长和饥饿生存的全基因组定量遗传分析

• 批准号: 8759128
• 负责人: Larry Ryan Baugh
• 金额: $ 19.08万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-10 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8759128
• 关键词: 5' -AMP-activated protein kinase; Adult; Affect; Aging; Alleles; American; Architecture; Area; Autophagocytosis; Biological Models; Buffers; Caenorhabditis elegans; Candidate Disease Gene; DNA Repair; Data; Development; Diabetes Mellitus; Disease; Essential Genes; Famines; Fertility; Future; Gene Frequency; Gene-Modified; Generations; Genes; Genetic; Genetic Epistasis; Genetic Techniques; Genome; Goals; Growth; Homeostasis; Individual; Insertional Mutagenesis; Insulin; Knowledge; Larva; Longevity; Malignant Neoplasms; Measures; Mediating; Methodology; Methods; Mitochondria; Modeling; Molecular; Mutate; Mutation; Nematoda; Nutrient; Ontology; Outcome; Pathway interactions; Phenotype; Population; Protocols documentation; Publishing; Quantitative Genetics; RNA Interference; Reading; Research; Resistance; Respiration; Sampling; Signal Transduction; Starvation; System; Technology; Testing; Time; Transposase; Validation; Variant; Work; base; deep sequencing; design; gene conservation; genetic analysis; genome wide association study; genome-wide; innovation; interest; models and simulation; next generation sequencing; novel; novel diagnostics; novel strategies; novel therapeutics; positional cloning; public health relevance; research study; response; therapeutic target; trait

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.

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被全面鉴定，但已知的饥饿生存所必需的基因相对较少，而且只有少数能够提高生存率的基因。目前尚不清楚哪些途径是核心途径，哪些途径缺失，以及这些途径如何整合以对养分可用性产生协调反应。该项目的长期目标是确定抗饥饿性的遗传结构作为数量性状。这包括全面鉴定影响饥饿生存的基因及其成对上位相互作用。然而，现有的后生动物遗传分析技术相对费力且不定量，并且上位性分析方法通常侧重于个体遗传相互作用。我们正在开发一种大规模并行方法来测量基因组中每个基因对线虫感兴趣的表型的功能贡献。基本原理是使用转座子对大量群体进行插入诱变，选择感兴趣的表型（饥饿存活），并使用深序列转座子侧翼来测量等位基因频率。线虫是该项目的理想选择，因为线虫能够适应盛宴和饥荒的循环，有丰富的遗传工具包可用于这种创新方法，基因组紧凑，可以培养非常大的种群，并且具有必需的基因已被全面识别，有利于验证。我们使用的 Mos1 转座子是理想的，因为它在没有转座酶的情况下是稳定的，其突变率自然较低且可优化，并且采用了经典的正向遗传协议。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，并且有能力检测人群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，并全面识别增加或减少饥饿抵抗力的基因。我们假设，许多在传统筛选中没有明显表型的基因对于饥饿生存至关重要，但相对较少的基因会提高生存率。我们将通过以下三个具体目标来实现我们的目标：1) 通过在线虫测序 (Gen-Seq) 开发转座子介导的遗传学，2) 通过全面鉴定必需基因来验证 Gen-Seq，以及 3) 全面鉴定非必需基因改变饥饿抵抗力的基因。我们提出初步建模结果和协议设计以支持可行性。该提案具有创新性，利用下一代测序和模型系统遗传学的力量来开发一种将大大加快遗传分析速度的方法。由于饥饿生存的遗传基础尚未在后生动物中确定，因此结果将具有重要意义，尽管它是与疾病具有巨大相关性的基本特征。未来的工作将利用 Gen-Seq 对抗饥饿性背后的遗传相互作用进行全基因组鉴定。