Germline Stem Cell Analysis Tools

 描述(由申请者提供)：该项目将开发、优化和实施技术，以线性标记和/或遗传改变线虫中的单个生殖细胞。这种蠕虫中的生殖系与包括人类在内的所有动物的干细胞系统有许多共同的特征。例如，它是通过与产生配体的细胞微环境或利基相互作用来维持的，以激活生殖细胞上高度保守的Notch受体。这是第一个在细胞和分子水平上定义了一个生态位的干细胞系统。自那以后，这个干细胞系统已经成为一个强大的系统，可以理解干细胞在遗传、环境和生理变化时的扩张、静止和可塑性的动态。这种蠕虫的生命周期很短(3天)，是透明的，寿命很短，并且非常容易受到环境和遗传操作的影响。尽管有这些优势，但目前对生殖系干细胞系统的关键特征及其对环境和遗传扰动的反应的了解受到了限制，因为我们对细胞更新的机制以及它们在实验操作下是如何改变的了解相对较差。为了弥合这一差距，我们需要随着时间的推移跟踪单个干细胞及其细胞后代。这里提出的技术将弥合这一差距。利用线虫种系转基因表达的最新进展，来自其他生物的现有技术将被引入蠕虫。除了谱系标记之外，这项技术还将使操纵单个细胞或细胞亚群中的基因活动成为可能。这项技术对于这个模型系统来说是独一无二的，它将能够在个体的整个生命周期和整个动物生理扰动的条件下监测生殖系干细胞。这一进展将有利于更大的干细胞生物学和生殖系领域，因为在这个主要的模型系统中将可以访问更多的问题。

 DESCRIPTION (provided by applicant): This project will develop, optimize and implement technology to lineally mark and/or genetically alter individual germ cells in C. elegans. The germ line in this worm shares many features in common with stem cell systems in all animals, including humans. For example, it is maintained by virtue of interaction with a cellular microenvironment or niche that produces ligands to activate a highly conserved Notch receptor on the germ cells. It was the first stem cell system for which a niche was defined at the cell and molecular levels. This stem cell system has since emerged as a powerful system to understand the dynamics of stem cell expansion, quiescence and plasticity in response to genetic, environmental and physiological alterations. The worm has a short (3-day) lifecycle, is transparent, short-lived, and highly amenable to environmental and genetic manipulation. Despite these advantages, understanding critical features of the germline stem cell system and its responses to environmental and genetic perturbations is currently limited by our relatively poor understanding of cellular mechanisms of renewal and how they are altered under experimental manipulation. To bridge this gap, we need to follow individual stem cells and their cellular progeny over time. The technology proposed here will bridge this gap. Taking advantage of recent progress in germline transgene expression in C. elegans, existing technologies from other organisms will be brought to the worm. In addition to lineage marking, the technology will make possible the manipulation of gene activity in single cells or subsets of cells. Uniquely for this model system, the technology will enable monitoring of germline stem cells in individuals over their entire lifetime and in conditions of whole-animal physiological perturbations. This advance will benefit the greater stem cell biology and germline fields as additional questions will become accessible in this premier model system.