Embryonic stem (ES) cells are accessible for extensive genetic manipulations, therefore, providing a means to unveil functions of human genes. Our lab focuses on establishing new approaches for genetic analysis using ES cells. A major obstacle to performing recessive genetic screens in mammalian cells is the diploid nature of the genome. To overcome this barrier, we have been using an ES cell line that are Bloom- syndrome protein deficient (blm-/-). Bloom-deficient cells exhibit a much higher loss of heterozygosity than wild-type cells and can be used to generate homozygous recessive mutants required for screening. Taking advantage of this feature of Bloom deficient ES cells, we have devised a novel genetic screen to identify components of mammalian RNA interference (RNAi) pathway. RNAi is a recently discovered gene silencing phenomenon that occurs in a wide variety of organisms. RNAi has been implicated in a variety of biological and pathological processes, from normal embryonic developemnt to human cancers and neurodegenerative diseases. Because much of our understanding of RNAi pathway come from genetic and biochemical analysis in worms, plants and flies, a genetic screen in mouse ES cells will provide an exciting opportunity to examine the unique aspect of RNAi in a vertebrate system. In our design, RNAi competent blm-/- ES cells will be mutated using a retroviral gene trap that randomly integrates within the genome. The blm-/- cells create homozygous mutants for the retroviral integration events. We will then use drug selection to isolate putative RNAi mutants. To prove the principle of our design, we have successfully performed a small-scale screen and identified Argonaute 2, a known component of RNAi pathway and other putative RNAi defective mutant ES cell lines. These results have therefore validated our screen for novel RNAi components in mammalian cells. We propose to carry out a large scale screen for RNAi mutants using our established selection system in Bloom deficient ES cells. By completing a genome-wide recessive genetic screen in mammalian cells, we will not only have the unique opportunity to examine the RNAi pathway in the unexplored territory of the vertebrate genome, but also set an example for deciphering other genetic pathways using recessive genetic screens. ? ? ?
