High throughput genetic screens in mammalian cells

Genetic screening is a powerful tool for the discovery of gene function. Previously, mammalian genetics relied on mapping naturally occurring mutations, as few methods existed for generating loss-of-function mutations and recovering them. With the discovery of RNAi, methods were developed that allowed the inhibition of gene expression in mammalian tissue culture. These methods allow the genetic dissection of diseases such as cancer, which were not previously accessible to genetic analysis. The studies in this dissertation sought new methods of conducting genetic screens in mammalian cells, with an emphasis on screening for cancer-specific lethal genes. To conduct lethal screens in mammalian cells, we first developed microarray methods for tracking the abundance of short hairpin RNA (shRNA) clones in a population. Initial methods using the full shRNA sequence or a random 60mer barcode proved insufficient to conduct large scale screens, due to poor dynamic range. To address this problem, we developed a method of using half of the shRNA sequence as a microarray probe. These half-hairpin probes had greater dynamic range than previous methods, and allowed us to conduct the first large-scale shRNA screens for cancer-lethal genes in batch culture. These screens enabled the recovery of many cancer-specific lethal genes, which represent possible cancer therapeutic targets. To address the shortcomings in current RNAi technology, we also conducted experiments on probe hybridization, discovering new rules for microarray probe design. These new rules will permit the barcoding of future libraries with more optimal array probes. To improve future shRNA knockdown, we also conducted a screens for synthetic enhancer elements that were capable of driving stronger transcription than current promoters. These screens recovered novel enhancer elements stronger than the cytomegalovirus (CMV) enhancer, though they were too cell-line specific to be useful for most RNAi studies at this time. The synthetic enhancers recovered, however, provide valuable information about the transcriptional state of different cell lines, and may one day lead to the development of very strong promoter elements.