REVERSIBLE PROMOTER-INSERTION FOR IN VIVO STUDIES OF MELANOMA

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DESCRIPTION (provided by applicant): It is commonly accepted that the processes underlying the development of cancer are best modeled in vivo. A plethora of methods have been developed for genetic dissection of biological processes in somatic cells in culture; however, the adaptation of these techniques to in vivo use has been very limited. Our earlier experience indicates that reversible promoter-insertion mutagenesis offers a number of advantages over the currently used forward genetics techniques for cultured cells. The most important benefits are the dominant nature of the mutations, and the ease of identifying the affected gene and establishing the causal link between the mutation and the phenotype. The principal limitation for the adaptation of this technique to in vivo studies is the delivery of the insertional mutagen (typically, a retroviral vector) to the desired cell type. Based on our recent experience and published data from others, we propose a method to circumvent this limitation via the use of specially engineered transposon vectors. We propose to apply our technique to study the genetic events underlying melanoma development. We will screen for genetic events that cooperate with oncogenic Ras (commonly found in melanomas) in this process. We will confirm the properties of the proposed insertional mutagens in culture and then proceed to establish mouse strains suitable for mutagenesis. Upon verifying the properties of our constructs in vivo, we will selectively mutagenize melanocytes in transgenic animals and will test the functional link between tumorigenesis and the inserted promoter. Confirmed hits will be the subject of future investigation, while the technique itself will become available for dissection of various biological phenomena in general and aspects of tumorigenesis, in particular.