COPAS BIOSORT Worm Sorter

COPAS BIOSORT 蠕虫分选机

• 批准号: 7795521
• 负责人: WOLFGANG B. LIEDTKE
• 金额: $ 44.63万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-10 至 2010-12-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7795521
• 关键词: American Society of Hematology; Bacterial Infections; Biological; Caenorhabditis elegans; Calcium; Candidate Disease Gene; Cations; Cell Separation; Cells; Cloning; Defect; Development; Disease; Event; Fluorescence; Generations; Genes; Genetic; Genome; Genomics; Grant; Head; Immune response; Individual; Inflammatory Bowel Diseases; Life; Malignant Neoplasms; Minor; Modeling; Molecular; Morphogenesis; Multi-Drug Resistance; Mutate; Nematoda; Neural Tube Closure; Neurons; Nociception; Optics; Organism; Proteins; Reporter; Reporting; Research; Research Personnel; Role; Sepsis; Sorting - Cell Movement; Structure of trigeminal ganglion; Technology; Time; Transgenes; Trigeminal Neuralgia; Universities; Vertebrates; calcium indicator; congenital heart disorder; gastrulation; high throughput screening; human disease; instrument; mature animal; novel; promoter; quantum; response; sensory mechanism; tool

DESCRIPTION (provided by applicant): Caenorhabditis elegans, a multicellular roundworm of approximately 1000 differentiated cells in adult animals, offers numerous advantages, namely it has an extremely short generation time, and its genome can be readily mutagenized, so that together with readily available genetic and genomic tools, cloning of mutated genes is highly feasible and greatly facilitated. Another advantage is optic transparency of the organism and availability of numerous gene-specific promoters that direct transgenes such as fluorescent proteins to specific cells, including neurons. Fluorescent indicators can be simply reporting the presence of a cell by decorating it with fluorescence or by functioning as biological reporters, such as genetically-encoded calcium indicators for changes in compartmental (such as cytoplasmic) calcium. Sorting technology developed for fluorescent cell sorting has been applied for sorting of multicellular organisms of appropriate size, such as C. elegans. Here we propose an automated sorter for C. elegans, to be located at Duke University, Durham, NC, for use by a group of investigators at Duke University and neighboring UNC at Chapel Hill. Three major users will be investigating specific questions pertinent to their R01 grants with the proposed instrument, all will be separating individual live worms, in which a fluorescent transgene will be directed to a specific cell by a specific promoter. By harnessing the power of worm sorting to conduct high-throughput assaying, combined with forward genetics, which in turn will be greatly facilitated by the sorter, their respective research agendas will be amplified by a quantum leap, at that level not feasible without the instrument. Dr. Liedtke, the PI, will be using a mammalianized worm model to study transduction mechanisms of the mammalian TRPV4 cation channel directed to the ASH nociceptive head neuron, the cellular equivalent of the trigeminal ganglion of vertebrates, in order to study molecular mechanisms of sensory transduction in response to noxious osmotic stimulation. These studies will help elucidate the role of TRPV4 in trigeminal pain. Dr. Aballay, also of Duke, will be investigating molecular mechanisms of C. elegans' immune response to bacterial invasion. His studies will help elucidate immun-pathogenetic mechanisms operative in diseases such as inflammatory bowel disease, sepsis, and multi-drug resistant bacterial infections. Dr. Goldstein, of UNC Chapel Hill, will be studying molecular mechanisms of gastrulation, an early developmental event of all multicellular organisms, in particular a set of novel candidate genes discovered in his lab. His studies will increase our understanding of cellular and molecular mechanisms of early morphogenesis relating to human disease states such as neural tube closure defects, cancer, and congenital heart disease. A strong and diverse group of minor users' projects will be contributing and benefitting as well.

描述(申请人提供)：秀丽隐杆线虫是一种在成年动物体内由大约1000个分化细胞组成的多细胞蛔虫，具有许多优点，即它的世代时间极短，其基因组可以很容易地发生突变，因此，结合现成的遗传和基因组工具，克隆突变基因是非常可行和非常方便的。另一个优势是有机体的光学透明性和许多基因特异性启动子的可用性，这些启动子将转基因(如荧光蛋白)定向到特定的细胞，包括神经元。荧光指示剂可以简单地通过用荧光装饰细胞来报告细胞的存在，或者通过充当生物报告程序来报告细胞的存在，例如通过遗传编码的钙指示剂来指示隔室(如细胞质)钙的变化。为荧光细胞分选开发的分选技术已被应用于适当大小的多细胞生物的分选，例如线虫。在这里，我们建议将线虫的自动分选器放置在北卡罗来纳州达勒姆的杜克大学，供杜克大学和邻近的北卡罗来纳大学教堂山分拣中心的一组研究人员使用。三个主要用户将使用拟议的工具调查与他们的R01拨款相关的特定问题，所有这些都将分离单个活蠕虫，其中荧光转基因将由特定的启动子定向到特定的细胞。通过利用蠕虫分选的力量进行高通量分析，结合正向遗传学，这反过来将极大地促进分选器的工作，他们各自的研究议程将以量子飞跃的方式放大，在没有仪器的情况下，这一水平是不可行的。PI的Liedtke博士将使用哺乳动物蠕虫模型来研究哺乳动物TRPV4阳离子通道指向ASH伤害性头部神经元的转导机制，以研究感觉转导对伤害性渗透刺激的反应的分子机制。这些研究将有助于阐明TRPV4在三叉神经痛中的作用。同样来自杜克大学的阿巴莱博士将研究线虫对细菌入侵的免疫反应的分子机制。他的研究将有助于阐明在炎症性肠病、败血症和多重耐药细菌感染等疾病中操作的免疫致病机制。北卡罗来纳大学教堂山分校的戈尔茨坦博士将研究原肠形成的分子机制，原肠形成是所有多细胞生物体的早期发育事件，特别是在他的实验室发现的一组新的候选基因。他的研究将增加我们对早期形态发生的细胞和分子机制的理解，这些机制与人类疾病状态有关，如神经管闭合缺陷、癌症和先天性心脏病。一个强大和多样化的小用户项目群体也将做出贡献并从中受益。