Cell-Based Assays for High-Throughput Screening

O'Rourke EJ, Conery AL, Moy TI, Eyleen J. O’Rourke, Annie L. Conery, Terence I. Moy, O’Rourke, Eyleen J., Conery, Annie L., Moy, Terence I.

The nematode Caenorhabditis elegans shows a high degree of conservation of molecular pathways related to human disease, yet is only 1-mm long and can be considered as a microorganism. Because of the development of a simple but systematic RNA-interference (RNAi) methodology, C. elegans is the only metazoan in which the impact of "knocking-down" nearly every gene in the genome can be analyzed in a whole living animal. Both functional genomic studies and chemical screens can be carried out using C. elegans in vivo screens in a context that preserves intact cell-to-cell communication, neuroendocrine signaling, and every aspect of the animal's metabolism necessary to survive and reproduce in lab conditions. This feature enables studies that are impossible to undertake in cell-culture-based screens. Although genome-wide RNAi screens and limited small-molecule screens have been successfully performed in C. elegans, they are typically extremely labor-intensive. Furthermore, technical limitations have precluded quantitative measurements and time-resolved analyses.In this chapter, we provide detailed protocols to carry out automated high-throughput whole-animal RNAi and chemical screens. We describe methods to perform screens in solid and liquid media, in 96 and 384-well format, respectively. We describe the use of automated handling, sorting, and microscopy of worms. Finally, we give information about worm-adapted image analysis tools to quantify phenotypes. The technology presented here facilitates large-scale C. elegans genetic and chemical screens and it is expected to help shed light on relevant biological areas.