Establishment of C.elegans tissue culture cell lines

描述（由申请人提供）：线虫秀丽隐杆线虫是一个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，尚无秀丽隐杆线虫组织培养细胞系。秀丽隐杆线虫细胞系将允许许多目前无法获得的实验方法，包括：同质细胞群的生物化学；细胞周期同步用于特定细胞周期阶段的分子分析体外分化研究；细胞自主缺陷的大规模RNAi筛选；还有小分子抑制剂筛选。此外，生殖细胞系将为靶向基因替换提供基础。历史上有两个主要的障碍阻碍了秀丽隐杆线虫细胞系的产生。首先，所有秀丽隐杆线虫的体细胞都经历了发育程序化的细胞周期阻滞。其次，动物的生殖细胞是合胞的，不能作为单个细胞分离出来。使用特定的遗传背景使我们能够克服这两个最初的障碍。这一建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不分化的增殖状态，因为分化将细胞从增殖池中移除；而且生殖细胞必须在培养中被诱导分裂。将使用许多方法来获得细胞系，包括优化培养基（如使用秀丽隐杆线虫提取物和细胞饲养系统），确定适当的细胞密度进行培养，并确定细胞附着条件。不同的突变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阻滞。重组GLP-1配体将提供给培养中的生殖细胞以阻止减数分裂进入。另一种情况是，生殖细胞可能来源于突变体背景，这些突变体对GLP-1信号通路具有组成性活性，因此不会进入减数分裂。细胞的分化标记、核型和RNAi能力将被表征。秀丽隐杆线虫在理解细胞死亡、发育生物学、细胞周期和信号转导方面取得了突破，对癌症和人类遗传疾病具有重要意义。细胞系的可用性将极大地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.