Engineering the C. Elegans Genome

线虫基因组工程

• 批准号: 8963980
• 负责人: ERIK M JORGENSEN
• 金额: $ 26.82万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8963980
• 关键词: Adult; Aging; Alleles; Anatomy; Animal Model; Bioinformatics; Biological Assay; Biology; Brain; C. elegans genome; Caenorhabditis elegans; Cell Death; Cell Nucleus; Cells; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Color; Communities; Complement; Complementary DNA; Computer software; Development; Drosophila genus; Engineering; Equilibrium; FLP recombinase; Funding; Future; Gene Expression Profile; Gene Targeting; Generations; Genes; Genetic; Genetic Models; Genetic Recombination; Genetic Screening; Genetic Transcription; Genome; Grant; Health; Human; Image; Individual; Introns; Knock-out; Libraries; Malignant Neoplasms; Manuals; Messenger RNA; Methods; MicroRNAs; Microscope; Microscopy; Mitogen-Activated Protein Kinases; Mutate; Nematoda; Neurons; Neurosciences; Oligonucleotides; Operon; Organism; Pathway interactions; Pattern; Plasmids; Play; Proteins; RNA Interference Pathway; Regulation; Reporter; Reporting; Research; Research Personnel; Resources; Role; Site; System; Testing; Three-Dimensional Image; Tissues; Toxicology; Transcript; Transgenes; Translations; United States National Institutes of Health; arm; base; cell type; cost; density; design; design and construction; null mutation; open source; pathogen; promoter; public health relevance; response; screening; tool

 DESCRIPTION (provided by applicant): Unbiased forward genetic screens play a central role in model organism genetics. C. elegans screens have 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. However, only ~10% of genes have alleles that have been isolated in forward screens. We propose to develop a transposon-based gene trap that will simultaneously generate balanced knockout alleles and cellular expression patterns. As a complement to this method, we will design constructs and methods to standardize and lower the costs of CRISPR-based gene traps. Although C. elegans has possibly the most thoroughly characterized anatomy of any organism, worm genetics has been confounded by the difficulty of unambiguously assigning expression of a specific gene to a particular cell. We will generate worm strains and open-source microscope hardware that will aid individual users as well as automated systems in solving this final step in assigning gene expression patterns. As a package, the methods will propose to develop will be a resource for the whole C. elegans research community. Aim 1. Random gene traps. We will modify the Mos1 transposon from Drosophila so that it will act as a gene trap when inserted into the C. elegans genome, that is, disrupt the gene and report the gene expression pattern with tagRFP. Aim 2. Directed gene traps. We will design and test a high-throughput strategy for targeted gene traps using CRISPR. The library of clones generated in this aim can be also be used by individual labs to obtain the expression pattern and a null allele of any gene in the genome. Aim 3. Expression pattern toolkit. We will build nematode strains and microscopy hardware for high-throughput recording and analyzing the expression patterns of genes in 3D. These tools will be applied to the gene traps but will also be of use to the C. elegans community for cell expression identities, particularly in the brain of the worm.

 描述（由适用提供）：无偏向的前遗传筛选在模型生物遗传学中起着核心作用。秀丽隐杆线虫的筛查在过去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的转录后调控就是例子。但是，只有约10％的基因具有在正向屏幕中分离出来的等位基因。我们建议开发一个基于转座子的基因陷阱，该基因陷阱将简单地产生平衡的基因敲除等位基因和细胞表达模式。作为对这种方法的补充，我们将设计构造和方法，以标准化和降低基于CRISPR的基因陷阱的成本。尽管秀丽隐杆线虫可能是任何生物体中最彻底的解剖结构，但蠕虫遗传学已被明确分配的特定基因表达到特定细胞的表达的困难所困扰。我们将生成蠕虫菌株和开源显微镜硬件，该硬件将帮助单个用户以及自动化系统解决分配基因表达模式的最后一步。作为包装，这些方法将提出开发的方法，将是整个秀丽隐杆线虫研究社区的资源。目标1。随机基因陷阱。我们将从果蝇中修改MOS1转座子，以便将其插入秀丽隐杆线虫基因组时，即破坏基因并用TAGRFP报告基因表达模式。目标2。定向基因陷阱。我们将使用CRISPR设计和测试针对靶向基因陷阱的高通量策略。单个实验室也可以使用此目标中产生的克隆库来获得基因组中任何基因的表达模式和无效等位基因。目标3。表达模式工具包。我们将构建线虫菌株和显微镜硬件，用于高通量记录，并分析3D中基因的表达模式。这些工具将应用于基因陷阱，但也将用于秀丽隐杆线虫社区的细胞表达身份，尤其是在蠕虫的大脑中。