Project description:Definition using LAM-PCR of the insertion profile of IFP2 transposons containing a NeoR using the wild type CRD-less PB transposase (1-558)
Project description:Definition of the insertion profile of Tcr-pble transposons containing a NeoR using G401 cells expressing PGBD5 Hs524 using LAM-PCR.
Project description:Definition using LAM-PCR of the insertion profile of IFP2 transposons containing a NeoR using the wild type CRD-less PB transposase (1-558) N-terminally fused with a SV40 NLS
Project description:Genomic rearrangements are a hallmark of childhood cancers, but the mutational processes underlying most of these variants remain unknown. We identified piggyBac transposable element derived 5 (PGBD5) as a highly expressed, enzymatically active domesticated human DNA transposase in a large subset of pediatric solid tumors, including rhabdoid tumors. Ectopic expression of PGBD5 in primary human cells was sufficient to induce fully penetrant cell transformation both in vitro and in immunodeficient mice in vivo. This activity required specific catalytic aspartic acid residues in the PGBD5 transposase domain as well as cellular non-homologous end-joining DNA repair, and was associated with distinct structural rearrangements defined by specific DNA sequence motifs. Similar genomic alterations, some recurrent, were found in primary human rhabdoid tumors. Thus, PGBD5 represents a new class of developmental oncogenic mutators in childhood solid tumors.
Project description:Induction of somatic oncogenic mutations by the domesticated DNA transposase PGBD5 in cerebellar progenitor cells promotes medulloblastoma development.
Project description:Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Retroviruses and transposons have been separately used as somatic cell insertional mutagens to identify cancer drivers in model organisms. Here we combined these mutagenic elements to enable cancer gene discovery starting with normal human cells. Lentivirus was used to seed gain- and loss-of-function gene disruption elements which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors rapidly generated in this context were high-grade sarcomas corresponding to the spectrum of myxofibrosarcoma and undifferentiated pleomorphic sarcoma, aggressive neoplasms with a predilection for older adults. Tumor insertion sites were genome-wide and enriched in regions of recurrent somatic copy number alteration found in multiple cancers, with a bias towards those of sarcomas. Novel driver genes which sustain somatic alterations in cancer patients were pinpointed. We identify the gene HDLBP, which codes for the RNA binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one in ten tumors across multiple tissues of origin. Hybrid viral-transposon systems will accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis. Lentivirus was used to seed gain- and loss-of-function gene disruption elements which were further deployed by Sleeping Beauty (SB) transposons throughout the genome of human bone explant mesenchymal cells. Genomic locations of LV (lentiviral backbone) and SB insertion sites were mapped by a pooled strategy utilizing linear amplification mediated PCR (LAM-PCR), followed by Illumina next generation sequencing of the product pool.
Project description:We describe a novel method of mapping genome-wide distributions of epigenetic marks in single cells using a Tn5 transposase complex conjugated to Protein A. This construct is guided to chromatin by an associated antibody, allowing sequence tag insertion and chromatin fragmentation specifically at genomic sites presenting the relevant antigen. This method is capable of processing thousands of individual cells in a single day of bench work.