Cancer gene discovery using somatic transposon mutagenesis in the mouse and systems for validation of identified candidate cancer genes and pathways.

Abstract

The conditional Sleeping Beauty (SB) transposon mutagenesis system has proven to be a successful method for cancer gene identification in solid tumors. Using tissue specific Cre recombinases (TSP-Cre) to selectively activate SB mediated mutagenesis in tissues of interest has yielded numerous novel candidate cancer genes in leukemia/lymphoma, colorectal, liver, and pancreatic cancers. We implemented the SB system for the identification of genes involved in osteosarcoma (OS). OS is the most common cancer of the bone and third most common cancer in children and adolescents. The 5-year survival rate of OS patients is 60-70%, though with metastatic disease it drops to less than 20%-30%. Identification of genes responsible for OS development and metastasis has been difficult due to its genomic instability and subsequent complexity. Consequently, there is a pressing need to identify the genes and pathways governing OS development and metastasis. To this end, we performed a forward genetic screen utilizing the conditional Sleeping Beauty (SB) transposase and mutagenic T2/Onc transposon system in Osx-Cre mice on a predisposing Trp53LSL-R270H/+ or wild type background to induce random somatic mutations that induce OS development and metastasis. One hundred and nineteen OSs were isolated from 96 predisposed and 20 wild type mice undergoing SB mutagenesis, with a subset of animals harboring metastases to the liver and/or lungs. Over 100 candidate cancer genes, both known and novel, were identified by analysis of recurrent T2/Onc insertion sites. In order to validate the candidate cancer genes identified in our screen, we developed a recombinase-based system for efficient assembly of vectors to over express or knock down 1-6 genes in mammalian cells. Further, these vectors were constructed in Piggybac transposons to allow for stable and reversible integration of the vector via transposition. In addition to cDNA over expression or shRNA knock down, we also developed methods for generation and selection of candidate cancer gene knock out cell lines using TAL effector nucleases (TALENs).