Caenorhabditis elegans, a multicellular roundworm of approximately 1000 differentiated cells in adult animals, offers numerous advantages, namely it has an extremely short generation time, and its genome can be readily mutagenized, so that together with readily available genetic and genomic tools, cloning of mutated genes is highly feasible and greatly facilitated. Another advantage is optic transparency of the organism and availability of numerous gene-specific promoters that direct transgenes such as fluorescent proteins to specific cells, including neurons. Fluorescent indicators can be simply reporting the presence of a cell by decorating it with fluorescence or by functioning as biological reporters, such as genetically-encoded calcium indicators for changes in compartmental (such as cytoplasmic) calcium. Sorting technology developed for fluorescent cell sorting has been applied for sorting of multicellular organisms of appropriate size, such as C. elegans. Here we propose an automated sorter for C. elegans, to be located at Duke University, Durham, NC, for use by a group of investigators at Duke University and neighboring UNC at Chapel Hill. Three major users will be investigating specific questions pertinent to their R01 grants with the proposed instrument, all will be separating individual live worms, in which a fluorescent transgene will be directed to a specific cell by a specific promoter. By harnessing the power of worm sorting to conduct high-throughput assaying, combined with forward genetics, which in turn will be greatly facilitated by the sorter, their respective research agendas will be amplified by a quantum leap, at that level not feasible without the instrument. Dr. Liedtke, the PI, will be using a mammalianized worm model to study transduction mechanisms of the mammalian TRPV4 cation channel directed to the ASH nociceptive head neuron, the cellular equivalent of the trigeminal ganglion of vertebrates, in order to study molecular mechanisms of sensory transduction in response to noxious osmotic stimulation. These studies will help elucidate the role of TRPV4 in trigeminal pain. Dr. Aballay, also of Duke, will be investigating molecular mechanisms of C. elegans'immune response to bacterial invasion. His studies will help elucidate immun-pathogenetic mechanisms operative in diseases such as inflammatory bowel disease, sepsis, and multi-drug resistant bacterial infections. Dr. Goldstein, of UNC Chapel Hill, will be studying molecular mechanisms of gastrulation, an early developmental event of all multicellular organisms, in particular a set of novel candidate genes discovered in his lab. His studies will increase our understanding of cellular and molecular mechanisms of early morphogenesis relating to human disease states such as neural tube closure defects, cancer, and congenital heart disease. A strong and diverse group of minor users'projects will be contributing and benefitting as well.
