title: High-throughput yeast plasmid overexpression screen.
abstract: The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology (1-5). In the last few years, several genomewide libraries of yeast strains and plasmids have been generated (6-10). These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches (11-16). Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.
authors.affiliation: Neuroscience Graduate Group, University of Pennsylvania School of Medicine, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Fleming
authors.forename: Aaron D
authors.initials: AD
authors.lastname: Gitler
mesh_terms: D010957:Plasmids / Q000235:genetics*; D012371:Robotics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic