Culture of differentiated C elegans embryonic cells

描述(由申请人提供)： 线虫为线虫提供了实质性的实验优势 离子通道-和的分子/遗传学基础研究 转运蛋白介导的细胞和整个动物的整合生理 流程。这些优势包括全基因组测序，寿命短 周期、遗传可控性和分子可操纵性。 然而，线虫在膜研究中的一个显著限制 运输现象是直接生理的相对不可及的 已分化的体细胞的测量。缺乏可靠的方法来解决 分化线虫细胞类型的原代或连续培养 对许多重要的生理学研究构成了额外的限制 和细胞生物学过程。最近，我们定义了C. 秀丽的胚胎细胞在培养中发育并存活数天至数周。 培养的胚胎细胞经历了惊人的分化形成神经元， 肌肉细胞和上皮型细胞。培养中的细胞可以很容易地贴片 夹紧并装载离子敏感荧光染料以供研究 定量成像方法。培养细胞中的靶基因功能是 通过在培养基中添加双链RNA而被破坏。组合直接 生理测量具有直截了当、强有力和高度选择性的特点 干扰基因表达的方法为 在分子水平上定义复杂的细胞过程。中心目标 这一建议的重点是进一步开发和表征C. 雅致的原代细胞培养。具体地说，我们将开发FACS方法来 纯化特定的绿色荧光蛋白标记的细胞类型。FACS方法将允许蜂窝 生化分析，基因组和蛋白质组谱，以及共培养 相互作用的细胞类型。我们还将进一步在体外开发RNA介导的基因 干扰策略，并进行初步的功能表征 培养线虫上皮细胞。考虑到相对容易和经济的 体内操纵线虫基因功能的原代细胞培养方法 使得很容易定义细胞生理学的遗传基础成为可能 过程，并将它们整合到整个动物的背景中。速度 而在线虫身上可以做到这一点的经济性是无法复制的 其他后生动物。

DESCRIPTION (provided by applicant): The nematode C. elegans offers substantial experimental advantages for investigations of the molecular/genetic basis of ion channel- and transporter-mediated cellular and whole animal integrated physiological processes. These advantages include a fully sequenced genome, a short life cycle, genetic tractability, and molecular manipulability. However, a significant limitation of C. elegans for studies of membrane transport phenomena is the relative inaccessibility for direct physiological measurements of differentiated somatic cells. The absence of robust methods for either primary or continuous culture of differentiated nematode cell types poses an additional limitation for studies of a host of important physiological and cell biological processes. Recently, we defined conditions in which C. elegans embryonic cells develop and survive in culture for many days to weeks. Cultured embryonic cells undergo striking differentiation to form neurons, muscle cells and epithelial-type cells. Cells in culture can be readily patch clamped and loaded with ion-sensitive fluorescent dyes for study by quantitative imaging methods. Targeted gene function in cultured cells is disrupted by adding double strand RNA to the culture medium. Combining direct physiological measurements with a straightforward, potent and highly selective method for disrupting gene expression offers extraordinary opportunities for defining complex cellular processes at the molecular level. The central goals of this proposal are focused on further development and characterization of C. elegans primary cell cultures. Specifically, we will develop FACS methods for purifying specific GFP-labeled cell types. FACS methods will allow cellular biochemical analysis, genomic and proteomic profiling, and co-culture of interacting cell types. We will also further develop in vitro RNA-mediated gene interference strategies and carry out an initial functional characterization of cultured nematode epithelial cells. Given the relative ease and economy of manipulating C. elegans gene function in vivo, primary cell culture methods now make it possible to readily define the genetic basis of cellular physiological processes and integrate them into the context of the whole animal. The speed and economy with which this can be done in C. elegans cannot be duplicated in other metazoan animals.