Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.
"PB Insertional Mutagenesis Screen in Human ES Cells The PB transposon has been demonstrated to be a useful tool for efficient transgenesis and insertional mutagenesis in both mouse and human immortalized cells (Ding et al., 2005). The transposon can efficiently mediate both loss-and gain-of-function insertional mutagenesis in mice (Ding et al., 2005; Rad et al., 2010; Landrette et al., 2011). Given that PB can also mediate efficient gene transfer in human ES cells (Chen et al., 2010), we decided to develop a PB vector for insertional mutagenesis screens in human ES cells. "
"A central problem in developmental biology is to identify the network of genes underlying the formation, migration, and assembly of these cells into the heart muscle tissue, the pacemaker and conduction system, and the coronary vasculature . Previously, genome-wide RNA interference (RNAi) loss-of-function screenings have provided a wealth of information in diverse model systems (Berns et al. 2004; Boutros et al. 2004; Carette et al. 2009; Jiang et al. 2009; Rad et al. 2010). Recent advances in CRISPR/Cas9 genome-editing technology and the cardiac differentiation method are now allowing genomewide loss-of-function screenings in human cells. "
[Show abstract][Hide abstract] ABSTRACT: Cardiogenesis is one of the earliest and most important steps during human development and is orchestrated by discrete families of heart progenitors, which build distinct regions of the fetal heart. For the past decade, a lineage map for the distinct subsets of progenitors that generate the embryonic mammalian heart has begun to lay a foundation for the development of new strategies for rebuilding the adult heart after injury, an unmet clinical need for the vast majority of patients with end-stage heart failure who are not heart transplant recipients. The studies also have implications for the root causes of congenital heart disease, which affects 1 in 50 live births, the most prevalent malformations in children. Although much of this insight has been generated in murine models, it is becoming increasingly clear that there can be important divergence with principles and pathways for human cardiogenesis, as well as for regenerative pathways. The development of human stem cell models, coupled with recent advances in genome editing with RNA-guided endonucleases, offer a new approach for the primary study of human cardiogenesis. In addition, application of the technology to the in vivo setting in large animal models, including nonhuman primates, has opened the door to genome-edited large animal models of adult and congenital heart disease, as well as a detailed mechanistic dissection of the more diverse and complex set of progenitor families and pathways, which guide human cardiogenesis. Implications of this new technology for a new generation of human-based, genetically tractable systems are discussed, along with potential therapeutic applications.
Cold Spring Harbor Perspectives in Medicine 09/2014; 6(10). DOI:10.1101/cshperspect.a013920 · 9.47 Impact Factor
"Yet other transposons, like recently identified TcBuster (6), SPIN (7) and piggyBat (8) elements, may offer alternative functional properties and new possibilities. The biomedical applicability of SB and PB is documented in cancer gene discovery (9–11), production of transgenic animals including large animal models (12,13), generation of induced pluripotent stem cells (14,15) and gene therapy (16,17). With the engineering of new hyperactive transposases [SB100X (18) and hyPBase (19) for SB and PB systems, respectively], DNA transposon systems have gained levels of activity that may in some cellular systems compare with the activity of viral vectors. "
[Show abstract][Hide abstract] ABSTRACT: DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.
Nucleic Acids Research 11/2013; 42(4). DOI:10.1093/nar/gkt1163 · 9.11 Impact Factor
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