The study of cancer has focused primarily on the genetic changes required to transform normal human cells into malignant cancers. Accordingly, it is thought that normal human cells require approximately five mutations to transform into a cancer cell. However, the identity of the cell population(s) in which these transforming events occurs remains undefined for most human cancers. The terminally differentiated cells that constitute the majority of epithelial tissues, such as the epidermis, are continually replaced making it unlikely that they would accumulate the number of genetic hits required for tumor formation. Interestingly, somatic stem cells inherently possess some of the hallmark traits of cancer cells raising the possibility that tumors may arise from stem cells through fewer or perhaps even a single mutational event. However, direct evidence for this "cancer stem cell hypothesis" is lacking due to the difficulty of manipulating and mutagenizing stem cell populations. Recently, our lab demonstrated highly efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. By separating the transposase (PBase) from PB, we generated a bipartite system in which the presence of both components in a cell is necessary for transposition. Spatially regulated PB transposition can be achieved by expressing PBase from a tissue- specific promoter, making this system particularly amenable to targeting stem cell populations for mutagenesis. The overall aim of this proposal is to use PB insertional mutagenesis to mutate epidermal stem cells, screen for mutations that promote tumorigenesis, and identify the affected genes. We will accomplish this as follows: (1) We will mutagenize cultured epidermal keratinocytes from both human and mouse and screen for in vitro malignant transformation utilizing a cell transformation assay for anchorage independent growth. (2) We will mutagenize epidermal stem cells in vivo by expressing transgenic PBase specifically in these cells to mobilize PB in mice. These mice will then be screened for skin tumor formation. Results from this study will allow direct evaluation of the cancer stem cell hypothesis as well as facilitate the identification of genes and pathways important in the initiation and progression of cancer. Relevance: This proposal seeks to develop new research methods that will allow for the identification of genes important for many human diseases. Specifically, results from this study will facilitate the identification of new genes involved in the initiation of human cancers. This information will increase the general understanding of how cancer develops as well as identify potential targets for cancer therapy.