Establishment of C.elegans tissue culture cell lines

描述（由申请人提供）：线虫C.线虫是一种强大的分子遗传系统，已被用于解决细胞、分子和发育生物学的基本问题。目前没有C。线虫组织培养细胞系。C. elegans细胞系将允许许多目前不可用的实验方法，包括：同质细胞群的生物化学;用于特定细胞周期阶段的分子分析的细胞周期同步化;体外分化研究;细胞自主缺陷的大规模RNAi筛选;以及小分子抑制剂筛选。此外，生殖细胞系将为靶向基因替代提供基础。历史上有两个主要的障碍阻碍了C语言的创建。elegans细胞系。首先，所有C。线虫体细胞经历发育程序性细胞周期停滞。第二，动物的生殖细胞是合胞体，不能分离为单个细胞。使用特定的遗传背景使我们能够克服这两个最初的障碍。该提议的重点是解决创建细胞系的剩余障碍：胚胎细胞必须保持在增殖状态而不分化，因为分化将细胞从增殖池中移除;并且必须诱导生殖细胞在培养中分裂。许多方法将用于获得细胞系，包括优化培养基（如使用C。elegans提取物和细胞饲养系统），确定用于培养的适当细胞密度，并鉴定细胞附着条件。不同的突变体背景将用于增加增殖和/或限制分化。端粒酶将在胚胎细胞中表达以对抗潜在的端粒缩短诱导的细胞周期停滞。将向培养的生殖细胞提供重组GLP-1配体，以阻断减数分裂进入。或者，生殖细胞将来源于对GLP-1信号传导途径具有组成型活性的突变体背景，因此从不进入减数分裂。将表征细胞的分化标志物、核型和RNAi能力。线虫C.线虫在细胞死亡、发育生物学、细胞周期和信号转导等方面的研究取得了突破性进展，这些研究对癌症和人类遗传疾病具有重要意义。细胞系的可用性将大大扩展使用这种生物体的研究，以提供进一步的健康相关见解。

DESCRIPTION (provided by applicant): The nematode C. elegans is a powerful molecular genetic system that has been used to address fundamental questions of cell, molecular, and developmental biology. Currently, there are no C. elegans tissue culture cell lines. C. elegans cell lines would allow a number of experimental approaches that are currently unavailable, including: biochemistry with homogenous cell populations; cell cycle synchronization for molecular analysis in specific cell cycle stages; studies of in vitro differentiation; large-scale RNAi screens for cell autonomous defects; and small molecule inhibitor screens. Additionally, a germ cell line would provide a foundation for targeted gene replacement. There are two major impediments that have historically blocked the creation of C. elegans cell lines. First, all C. elegans somatic cells undergo developmentally- programmed cell cycle arrest. Second, germ cells in the animal are syncytial and cannot be isolated as individual cells. The use of specific genetic backgrounds has allowed us to overcome both of these initial obstacles. This proposal is focused on addressing the remaining impediments to the creation of cell lines: embryonic cells must be maintained in a proliferative state without differentiating, as differentiation removes cells from the proliferative pool; and germ cells must be induced to divide in culture. A number of approaches will be used to attain cell lines, including optimizing the culture media (such as the use of C. elegans extract and cell feeder systems), defining the proper cell density for culturing, and identifying cell attachment conditions. Different mutant backgrounds will be used to increase proliferation and/or limit differentiation. Telomerase will be expressed in embryonic cells to counter potential telomere shortening- induced cell cycle arrest. Recombinant GLP-1 ligand will be provided to germ cells in culture to block meiotic entry. Alternatively, germ cells will be derived from mutant backgrounds that are constitutively active for the GLP-1 signaling pathway, and therefore never enter meiosis. Cells will be characterized for differentiation markers, karyotype, and RNAi capability. The nematode C. elegans has provided breakthroughs in the understanding of cell death, developmental biology, the cell cycle, and signal transduction that have had important implications for cancer and human genetic diseases. The availability of cell lines would greatly extend research using this organism to provide further health-related insights.