Automated single cell expression analysis in C. elegans

线虫中的自动单细胞表达分析

• 批准号: 7030533
• 负责人: ROBERT H WATERSTON
• 金额: $ 50.63万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030533
• 关键词: Caenorhabditis elegans; automated data processing; bioimaging /biomedical imaging; cell cycle; developmental genetics; embryo /fetus culture; fluorescent dye /probe; functional /structural genomics; genetic mapping; genetic promoter element; green fluorescent proteins; helminth genetics; histones; intravital microscopy; invertebrate embryology; mathematical model; method development; microorganism culture; population genetics; protein protein interaction; videotape /videodisc

DESCRIPTION (provided by applicant): The information stored in an organism's genome directs its development and behavior, but how that occurs is only beginning to be understood. Knowledge of what genes are used in each cell at every stage of development would be a significant step toward a comprehensive understanding. We propose to develop methods that will exploit the invariant lineage of the nematode C. elegans to define gene expression patterns throughout development at the single cell level with high temporal resolution. This will be achieved with two parallel reporter systems. We will computationally track each nucleus through movement, division and death in 3D movies of developing embryos expressing histone-GFP fusions in each cell, and thus automatically determine the lineage for each cell and hence its identity. Simultaneously, we will detect a second-color reporter expressed under the control of a candidate transcriptional regulatory region. By mapping the resulting temporal and spatial expression patterns onto the embryonic lineage, we will identify the cells expressing the reporter. These methods will require the creation of new techniques and the modification of existing one to enable the generation of new worm strains, live-cell fluorescence imaging and pattern recognition in images. We will assess the accuracy of the method through multiple quality control tests and begin to develop a pipeline to allow systematic application of the methods to large numbers of genes. The completed system will be useful for a wide variety of functional genomics applications and with further adaptation could provide an avenue to comprehensive analysis of gene expression in C. elegans. Knowledge of gene expression patterns in C. elegans would not only provide insights into the role of these genes in development, but would suggest roles of homologous genes in health and disease in other animals, including humans.

描述（由申请人提供）：存储在生物体基因组中的信息指导其发育和行为，但这种情况是如何发生的才刚刚开始被理解。了解每个细胞在每个发育阶段使用哪些基因将是迈向全面理解的重要一步。我们建议开发方法，利用线虫的不变谱系。 线虫以高时间分辨率在单细胞水平上定义整个发育过程中的基因表达模式。这将通过两个并行报告系统来实现。我们将通过计算在每个细胞中表达组蛋白-GFP 融合体的发育胚胎的 3D 电影中的运动、分裂和死亡来跟踪每个细胞核，从而自动确定每个细胞的谱系及其身份。同时，我们将检测在候选转录调控区控制下表达的第二颜色报告基因。通过将所得的时间和空间表达模式映射到胚胎谱系上，我们将识别表达报告基因的细胞。这些方法需要创建新技术并对现有技术进行修改，以生成新的蠕虫菌株、活细胞荧光成像和图像模式识别。我们将通过多次质量控制测试来评估该方法的准确性，并开始开发一个管道，以便将该方法系统地应用于大量基因。完整的系统将可用于各种功能基因组学应用，并且通过进一步的调整可以为线虫基因表达的综合分析提供途径。了解秀丽隐杆线虫的基因表达模式不仅可以深入了解这些基因在发育中的作用，还可以表明同源基因在包括人类在内的其他动物的健康和疾病中的作用。