Engineering the C. elegans Genome

改造秀丽隐杆线虫基因组

基本信息

批准号：
8204712
负责人：
ERIK M JORGENSEN
金额：
$ 25.42万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8204712
关键词：
Aging Alleles Animal Model Animals Area Biological Biology C. elegans genome Caenorhabditis elegans Cell Death Cellular biology Chromosomal Insertion Chromosomes Code Codon Nucleotides Communities DNA DNA Transposable Elements Engineering Excision Frequencies Funding Gene Silencing Gene Targeting Gene-Modified Genes Genetic Engineering Genetic Models Genetic Recombination Genome Genome engineering Genomics Health Human Humulus In Situ Knock-out Malignant Neoplasms Mediating Methods MicroRNAs Mitogen-Activated Protein Kinases Modification Mus Mutagenesis Mutation Neurosciences Nucleic Acid Regulatory Sequences Pathway interactions Phenotype Plasmids Protocols documentation RNA Interference RNA Interference Pathway Reagent Regulation Research Research Personnel Resistance Resources Site Techniques Time Tissues Toxicology Transgenes Transgenic Animals Transgenic Organisms United States National Institutes of Health Yeasts arm base fly gene function gene replacement genetic analysis improved knockout gene pathogen public health relevance reconstitution response vector

项目摘要

DESCRIPTION (provided by applicant): Genetic analysis relies on the ability to introduce, eliminate or modify genes at will. Such techniques are advanced in genetic model organisms such as yeast, flies and mice, but are limited in the worm C. elegans, which is one of the most commonly used model organisms. In worms such methods are somewhat crude: gene knockouts are by random mutagenesis and restoring gene function is via multicopy extrachromosomal arrays or random gene integrations. Despite these limitations C. elegans research has contributed to some of the most important discoveries in biology in the last 35 years: Ras - MAP kinase pathways, cell death pathways, RNA interference and posttranscriptional regulation by microRNAs are examples. We propose to develop techniques to insert, delete or modify genes in the C. elegans genome. The techniques rely on mobilizing transposons to engineer the genome; the methods will be a resource for the whole C. elegans research community. Aim 1. Improved single copy insertion. We will characterize insertion sites on each chromosome and increase the efficiency of transgene insertions. We will also devise transient selection reagents that can be used in a wild-type background. Aim 2. Universal insertion sites. We will generate universal insertion sites on all chromosomes that will be compatible with a single targeting plasmid. This will substantially increase the versatility of the technique. Aim 3. Gene targeting. We will develop a strategy to manipulate genes in their endogenous context. This technique will allow researchers to engineer mutations, including knock-outs, without any extraneous DNA changes in the gene except the intended mutation. PUBLIC HEALTH RELEVANCE: C. elegans is one of the major model organisms for studies of cell biology; more than 300 C. elegans labs in the US currently receive funding from the NIH. These labs pursue projects studying genes involved in aging, cancer, toxicology, neuroscience, and response to pathogens. The ability to modify the C. elegans genome without limits will make experimentation much faster and results more reliable for studies of all aspects of human health.

描述（由申请人提供）：遗传分析依赖于随意引入，消除或修改基因的能力。这种技术是在诸如酵母，苍蝇和小鼠等遗传模型生物中采用的，但在蠕虫秀丽隐杆线虫中受到限制，这是最常用的模型生物之一。在蠕虫中，这种方法有些粗糙：基因敲除是通过随机诱变，而恢复基因功能是通过多拷贝外染色体外阵列或随机基因整合。尽管有这些局限性，秀丽隐杆线虫的研究在过去35年中为生物学中一些最重要的发现做出了贡献：RAS- MAP激酶途径，细胞死亡途径，RNA干扰和microRNA的转录后调节是例子。我们建议开发在秀丽隐杆线虫基因组中插入，删除或修改基因的技术。这些技术依靠动员转座子来设计基因组。这些方法将是整个秀丽隐杆线虫研究社区的资源。 AIM 1。改进的单复制插入。我们将表征每个染色体上的插入位点，并提高转基因插入的效率。我们还将设计可以在野生型背景中使用的瞬态选择试剂。目标2。通用插入站点。我们将在所有染色体上生成通用插入位点，这些染色体将与单个靶向质粒兼容。这将大大提高该技术的多功能性。目标3。基因靶向。我们将制定一种在内源性环境下操纵基因的策略。该技术将使研究人员能够设计包括淘汰赛在内的突变，而没有任何外科DNA的变化，除了预期的突变。公共卫生相关性：秀丽隐杆线虫是细胞生物学研究的主要模型生物之一；目前，美国有300多个C.秀丽隐杆线索实验室从NIH获得资金。这些实验室追求研究涉及衰老，癌症，毒理学，神经科学和对病原体反应的基因。在不限制的情况下修改秀丽隐杆线虫基因组的能力将使实验更快，并且对人类健康各个方面的研究更可靠。