Sleeping beauty: a novel cancer gene discovery tool

ArticleinHuman Molecular Genetics 15 Spec No 1(suppl 1):R75-9 · May 2006with5 Reads
DOI: 10.1093/hmg/ddl061 · Source: PubMed
Abstract
The National Cancer Institute and the National Human Genome Research Institute recently announced a 3-year 100-million-dollar pilot study to use large-scale resequencing of genes in human tumors to identify new cancer genes. The hope is that some of these genes can be used as drug targets for developing better therapeutics for treating cancer. Although this effort will identify new cancer genes, it could be made more efficient by preferentially resequencing genes identified as novel candidate cancer genes in animal models of cancer. Although retroviral insertional mutagenesis has proven to be an effective tool for identifying novel cancer genes in the mouse, these studies are limited by the fact that retroviral mutagenesis primarily induces hematopoietic and mammary cancer, but little else, while the majority of cancers affecting humans are solid tumors. Recently, two groups have shown that sleeping beauty (SB) transposon-based insertional mutagenesis can also identify novel candidate cancer genes in the mouse. Unlike retroviral infection, SB transposition can be controlled to mutagenize any target tissue and thus potentially induce many different kinds of cancer, including solid tumors. SB transposition in animal models of cancer could therefore greatly facilitate the identification of novel human cancer genes and the development of better cancer therapies.
    • "Among the ten genes, seven (CNTN4, CTNNA3, EPHA6, FOXO1, MAGI1, PTPRD and SMAD4) were The Protein Domain Landscape of Cancer-Type-Specific Somatic Mutations predicted to be tumor suppressors. Using transposon insertion positions, the Sleeping Beauty study [60] had annotated three of these seven genes as loss of function (while not suggesting an annotation for the remaining four). We also reported two potential oncogenes, USP25 and GNA13 (Table 4). "
    [Show abstract] [Hide abstract] ABSTRACT: Author Summary Extensive tumor genome sequencing has provided raw material to understand mutational processes and identify cancer-associated somatic variants. However, fundamental problems remain to: i) separate ‘driver’ from ‘passenger’ mutations, ii) further understand the functional mechanisms and consequences of driver mutations, and iii) identify the cancer types in which each driver mutation is relevant. Here we analyze whole-genome and exome tumor sequencing data from the perspective of protein domains—the basic structural and functional units of proteins. Exploring the cancer-type-specific landscape of domain mutations across 21 cancer types, we identify both cancer-type-specific mutated domains and mutational hotspots. Frequently-mutated domains were identified for oncoproteins for which the ‘mutational hotspot’ phenomenon owing to the relative rarity of gain-of-function mutations is well known, and also for tumor suppressor proteins, for which more uniformly distributed loss-of-function driver mutations are expected. A given gene product may be perturbed differently in different cancers. Indeed, we observed systematic shifts between cancer types of the positions at which mutations occur within a given protein. Both known and novel candidate driver mutations were retrieved. Novel cancer gene candidates significantly overlapped with orthogonal systematic cancer screen hits, supporting the power of this approach to identify cancer genes.
    Full-text · Article · Mar 2015
    • "The Sleeping Beauty (SB) is the first DNA transposon system that has been described to work in mammalian cells and is also the most widely used transposon system for mammalian transgenesis and mutagenesis (Takeda et al., 2007; Wang et al., 2008 ). For example , SB has been used to identify new cancer genes (Collier et al., 2005; Dupuy et al., 2006) and generate transgene animals (Kitada et al., 2009). However, SB transposition application is limited by its relatively low transposition efficiency and its nonspecific integration (Wu et al., 2006; Yant et al., 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: The PiggyBac (PB) transposon system is a non-viral DNA-transfer system in which a transposase directs integration of a PB transposon into a TTAA site in the genome. Transgenic expression of porcine CD163 is necessary and sufficient to confer non-permissive cells susceptible to infection with porcine reproductive and respiratory syndrome virus (PRRSV). Such permissive cells can be used as a tool for PRRSV cellular receptor and other studies. One of the problems in studying PRRSV is the lack of porcine cell lines. In this study, efficient transfection and expression of porcine CD163 in PK-15 cells by PB transposition was demonstrated. The stable PK-15(CD163) cell line was used in PRRSV infection assays. The data indicated that the average PB transgene copy number per genome was approximately ten. In line with previous literature the integration of PB into the genome had a bias toward the TTAA chromosomal site. The PK-15(CD163) cell line was susceptible to infection by different PRRSV strains and the virus grew to similar titers compared to the Marc-145 cell line. This simplification of PK-15(CD163) cell line production will provide a valuable tool to facilitate PRRSV cellular receptor studies and to accelerate existing vectors for PK-15 cell-based gene transfer and expression.
    Full-text · Article · Jul 2013
    • "This proved to be a viable strategy for transient transgenic RNAi knockdown in mouse embryos using Prdm16-specific shRNAs, although the specialized training and facilities necessary for working with these pathogens reduces its attractiveness as a universal tool for these studies. The SB system is also a tractable means to perform in vitro and in vivo transgenic studies of many kinds, including cancer modeling, gene trapping, generation of transgenic mouse lines and insertional mutagenesis [33,34,35,36]. Several factors have been shown to affect the transposition efficiency of SB transposons in vitro. "
    [Show abstract] [Hide abstract] ABSTRACT: RNA interference (RNAi) is a powerful strategy for studying the phenotypic consequences of reduced gene expression levels in model systems. To develop a method for the rapid characterization of the developmental consequences of gene dysregulation, we tested the use of RNAi for "transient transgenic" knockdown of mRNA in mouse embryos. These methods included lentiviral infection as well as transposition using the Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems. This approach can be useful for phenotypic validation of putative mutant loci, as we demonstrate by confirming that knockdown of Prdm16 phenocopies the ENU-induced cleft palate (CP) mutant, csp1. This strategy is attractive as an alternative to gene targeting in embryonic stem cells, as it is simple and yields phenotypic information in a matter of weeks. Of the three methodologies tested, the PB transposon system produced high numbers of transgenic embryos with the expected phenotype, demonstrating its utility as a screening method.
    Full-text · Article · Dec 2010
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