Culture of differentiated C elegans embryonic cells

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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.