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. [unreadable] [unreadable]
NIH ExPorter
NIH ExPorter