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Transposon

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Transposon - Free Register to Access Experts & Abstracts

Zsuzsanna Izsvák - One of the best experts on this subject based on the ideXlab platform.

  • Transposon-mediated genome manipulation in vertebrates
    Nature Methods, 2009
    Co-Authors: Zoltán Ivics, Jef D Boeke, Lajos Mátés, Andras Nagy, Allan Bradley, Zsuzsanna Izsvák
    Abstract:

    Transposable elements are DNA segments with the unique ability to move about in the genome. This inherent feature can be exploited to harness these elements as gene vectors for genome manipulation. Transposon-based genetic strategies have been established in vertebrate species over the last decade, and current progress in this field suggests that transposable elements will serve as indispensable tools. In particular, Transposons can be applied as vectors for somatic and germline transgenesis, and as insertional mutagens in both loss-of-function and gain-of-function forward mutagenesis screens. In addition, Transposons will gain importance in future cell-based clinical applications, including nonviral gene transfer into stem cells and the rapidly developing field of induced pluripotent stem cells. Here we provide an overview of Transposon-based methods used in vertebrate model organisms with an emphasis on the mouse system and highlight the most important considerations concerning genetic applications of the Transposon systems.

  • sleeping beauty a wide host range Transposon vector for genetic transformation in vertebrates
    Journal of Molecular Biology, 2000
    Co-Authors: Zoltán Ivics, Zsuzsanna Izsvák, Ronald H A Plasterk
    Abstract:

    Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of transposable elements, is the only active DNA-based Transposon system of vertebrate origin that is available for experimental manipulation. We have been using the SB element as a research tool to investigate some of the cis and trans-requirements of element mobilization, and mechanisms that regulate transposition in vertebrate species. In contrast to mariner Transposons, which are regulated by overexpression inhibition, the frequency of SB transposition was found to be roughly proportional to the amount of transposase present in cells. Unlike Tc1 and mariner elements, SB contains two binding sites within each of its terminal inverted repeats, and we found that the presence of both of these sites is a strict requirement for mobilization. In addition to the size of the Transposon itself, the length as well as sequence of the DNA outside the Transposon have significant effects on transposition. As a general rule, the closer the Transposon ends are, the more efficient transposition is from a donor molecule. We have found that SB can transform a wide range of vertebrate cells from fish to human. However, the efficiency and precision of transposition varied significantly among cell lines, suggesting potential involvement of host factors in SB transposition. A positive-negative selection assay was devised to enrich populations of cells harboring inserted Transposons in their chromosomes. Using this assay, of the order of 10,000 independent Transposon insertions can be generated in human cells in a single transfection experiment. Sleeping Beauty can be a powerful alternative to other vectors that are currently used for the production of transgenic animals and for human gene therapy.

  • molecular reconstruction of sleeping beauty a tc1 like Transposon from fish and its transposition in human cells
    Cell, 1997
    Co-Authors: Zoltán Ivics, Zsuzsanna Izsvák, Perry B Hackett, Ronald H A Plasterk
    Abstract:

    Members of the Tc1/mariner superfamily of Transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to construct a synthetic Transposon, Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations. Sleeping Beauty transposase binds to the inverted repeats of salmonid Transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells. Sleeping Beauty is an active DNA-Transposon system from vertebrates for genetic transformation and insertional mutagenesis.

Ronald H A Plasterk - One of the best experts on this subject based on the ideXlab platform.

  • regulated transposition of a fish Transposon in the mouse germ line
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Sylvia E J Fischer, Erno Wienholds, Ronald H A Plasterk
    Abstract:

    Tc1/mariner elements are able to transpose in species other than the host from which they were isolated. As potential vectors for insertional mutagenesis and transgenesis of the mouse, these cut-and-paste Transposons were tested for their ability to transpose in the mouse germ line. First, the levels of activity of several Tc1/mariner elements in mammalian cells were compared; the reconstructed fish Transposon Sleeping Beauty (SB) was found to be an order of magnitude more efficient than the other tested Transposons. SB then was introduced into the mouse germ line as a two-component system: one transgene for the expression of the transposase in the male germ line and a second transgene carrying a modified Transposon. In 20% of the progeny of double transgenic male mice the Transposon had jumped from the original chromosomal position into another locus. Analysis of the integration sites shows that these jumps indeed occurred through the action of SB transposase, and that SB has a strong preference for intrachromosomal transposition. Analysis of the excision sites suggests that double-strand breaks in haploid spermatids are repaired via nonhomologous end joining. The SB system may be a powerful tool for Transposon mutagenesis of the mouse germ line.

  • sleeping beauty a wide host range Transposon vector for genetic transformation in vertebrates
    Journal of Molecular Biology, 2000
    Co-Authors: Zoltán Ivics, Zsuzsanna Izsvák, Ronald H A Plasterk
    Abstract:

    Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of transposable elements, is the only active DNA-based Transposon system of vertebrate origin that is available for experimental manipulation. We have been using the SB element as a research tool to investigate some of the cis and trans-requirements of element mobilization, and mechanisms that regulate transposition in vertebrate species. In contrast to mariner Transposons, which are regulated by overexpression inhibition, the frequency of SB transposition was found to be roughly proportional to the amount of transposase present in cells. Unlike Tc1 and mariner elements, SB contains two binding sites within each of its terminal inverted repeats, and we found that the presence of both of these sites is a strict requirement for mobilization. In addition to the size of the Transposon itself, the length as well as sequence of the DNA outside the Transposon have significant effects on transposition. As a general rule, the closer the Transposon ends are, the more efficient transposition is from a donor molecule. We have found that SB can transform a wide range of vertebrate cells from fish to human. However, the efficiency and precision of transposition varied significantly among cell lines, suggesting potential involvement of host factors in SB transposition. A positive-negative selection assay was devised to enrich populations of cells harboring inserted Transposons in their chromosomes. Using this assay, of the order of 10,000 independent Transposon insertions can be generated in human cells in a single transfection experiment. Sleeping Beauty can be a powerful alternative to other vectors that are currently used for the production of transgenic animals and for human gene therapy.

  • mut 7 of c elegans required for Transposon silencing and rna interference is a homolog of werner syndrome helicase and rnased
    Cell, 1999
    Co-Authors: Rene F Ketting, Thomas H A Haverkamp, Henri G A M Van Luenen, Ronald H A Plasterk
    Abstract:

    Abstract While all known natural isolates of C. elegans contain multiple copies of the Tc1 Transposon, which are active in the soma, Tc1 transposition is fully silenced in the germline of many strains. We mutagenized one such silenced strain and isolated mutants in which Tc1 had been activated in the germline ("mutators"). Interestingly, many other Transposons of unrelated sequence had also become active. Most of these mutants are resistant to RNA interference (RNAi). We found one of the mutated genes, mut-7 , to encode a protein with homology to RNaseD. This provides support for the notion that RNAi works by dsRNA-directed, enzymatic RNA degradation. We propose a model in which MUT-7, guided by Transposon-derived dsRNA, represses transposition by degrading Transposon-specific messengers, thus preventing transposase production and transposition.

  • molecular reconstruction of sleeping beauty a tc1 like Transposon from fish and its transposition in human cells
    Cell, 1997
    Co-Authors: Zoltán Ivics, Zsuzsanna Izsvák, Perry B Hackett, Ronald H A Plasterk
    Abstract:

    Members of the Tc1/mariner superfamily of Transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to construct a synthetic Transposon, Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations. Sleeping Beauty transposase binds to the inverted repeats of salmonid Transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells. Sleeping Beauty is an active DNA-Transposon system from vertebrates for genetic transformation and insertional mutagenesis.

Jim Leebensmack - One of the best experts on this subject based on the ideXlab platform.

  • hybridization history and repetitive element content in the genome of a homoploid hybrid yucca gloriosa asparagaceae
    Frontiers in Plant Science, 2021
    Co-Authors: Karolina Heyduk, Edward V Mcassey, Jane Grimwood, Shengqiang Shu, Jeremy Schmutz, Michael R Mckain, Jim Leebensmack
    Abstract:

    Hybridization in plants results in phenotypic and genotypic perturbations that can have dramatic effects on hybrid physiology, ecology, and overall fitness. Hybridization can also perturb epigenetic control of transposable elements, resulting in their proliferation. Understanding the mechanisms that maintain genomic integrity after hybridization is often confounded by changes in ploidy that occur in hybrid plant species. Homoploid hybrid species, which have no change in chromosome number relative to their parents, offer an opportunity to study the genomic consequences of hybridization in the absence of change in ploidy. Yucca gloriosa (Asparagaceae) is a young homoploid hybrid species, resulting from a cross between Yucca aloifolia and Yucca filamentosa. Previous analyses of ~11kb of the chloroplast genome and nuclear-encoded microsatellites implicated a single Y. aloifolia genotype as the maternal parent of Y. gloriosa. Using whole genome resequencing, we assembled chloroplast genomes from 41 accessions of all three species to re-assess the hybrid origins of Y. gloriosa. We further used re-sequencing data to annotate Transposon abundance in the three species and mRNA-seq to analyze transcription of Transposons. The chloroplast phylogeny and haplotype analysis suggest multiple hybridization events contributing to the origin of Y. gloriosa, with both parental species acting as the maternal donor. Transposon abundance at the superfamily level was significantly different between the three species; the hybrid was frequently intermediate to the parental species in TE superfamily abundance or appeared more similar to one or the other parent. In only one case - Copia LTR Transposons - did Y. gloriosa have a significantly higher abundance relative to either parent. Expression patterns across the three species showed little increased transcriptional activity of Transposons, suggesting that either no Transposon release occurred in Y. gloriosa upon hybridization, or that any Transposons that were activated via hybridization were rapidly silenced. The identification and quantification of Transposon families paired with expression evidence paves the way for additional work seeking to link epigenetics with the important trait variation seen in this homoploid hybrid system.

  • hybridization history and repetitive element content in the genome of a homoploid hybrid yucca gloriosa asparagaceae
    bioRxiv, 2020
    Co-Authors: Karolina Heyduk, Edward V Mcassey, Jane Grimwood, Shengqiang Shu, Jeremy Schmutz, Michael R Mckain, Jim Leebensmack
    Abstract:

    Abstract Hybridization in plants results in phenotypic and genotypic perturbations that can have dramatic effects on hybrid physiology, ecology, and overall fitness. Hybridization can also perturb epigenetic control of transposable elements, resulting in their proliferation. Understanding the mechanisms that maintain genomic integrity after hybridization is often confounded by changes in ploidy that occur in hybrid plant species. Homoploid hybrid species, which have no change in chromosome number relative to their parents, offer an opportunity to study the genomic consequences of hybridization in the absence of change in ploidy. Yucca gloriosa (Asparagaceae) is a young homoploid hybrid species, resulting from a cross between Yucca aloifolia and Yucca filamentosa. Previous analyses of ~11kb of the chloroplast genome and nuclear-encoded microsatellites implicated a single Y. aloifolia genotype as the maternal parent of Y. gloriosa. Using whole genome resequencing, we assembled chloroplast genomes from multiple accessions of all three species to re-assess the hybrid origins of Y. gloriosa. We further used re-sequencing data to annotate Transposon abundance in the three species and mRNA-seq to analyze transcription of Transposons. The chloroplast phylogeny and haplotype analysis suggest multiple hybridization events contributing to the origin of Y. gloriosa, with both parental species acting as the maternal donor. Transposon abundance at the superfamily level was significantly different between the three species; the hybrid was frequently intermediate to the parental species in TE superfamily abundance or appeared more similar to one or the other parent. In only one case – Copia LTR Transposons – did Y. gloriosa have a significantly higher abundance relative to either parent. Expression patterns across the three species showed little increased transcriptional activity of Transposons, suggesting that either no Transposon release occurred in Y. gloriosa upon hybridization, or that any Transposons that were activated via hybridization were rapidly silenced. Further work will assess the degree to which Transposon abundance and location has affected the epigenomic landscape, gene expression, and ecophysiology in Y. gloriosa.

Allan Bradley - One of the best experts on this subject based on the ideXlab platform.

  • Transposon-mediated genome manipulation in vertebrates
    Nature Methods, 2009
    Co-Authors: Zoltán Ivics, Jef D Boeke, Lajos Mátés, Andras Nagy, Allan Bradley, Zsuzsanna Izsvák
    Abstract:

    Transposable elements are DNA segments with the unique ability to move about in the genome. This inherent feature can be exploited to harness these elements as gene vectors for genome manipulation. Transposon-based genetic strategies have been established in vertebrate species over the last decade, and current progress in this field suggests that transposable elements will serve as indispensable tools. In particular, Transposons can be applied as vectors for somatic and germline transgenesis, and as insertional mutagens in both loss-of-function and gain-of-function forward mutagenesis screens. In addition, Transposons will gain importance in future cell-based clinical applications, including nonviral gene transfer into stem cells and the rapidly developing field of induced pluripotent stem cells. Here we provide an overview of Transposon-based methods used in vertebrate model organisms with an emphasis on the mouse system and highlight the most important considerations concerning genetic applications of the Transposon systems.

  • generation of transgene free induced pluripotent mouse stem cells by the piggybac Transposon
    Nature Methods, 2009
    Co-Authors: Kosuke Yusa, Roland Rad, Junji Takeda, Allan Bradley
    Abstract:

    Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac Transposon-based approach to generate integration-free iPSCs. Transposons carrying 2A peptide-linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed Transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac Transposon-based reprogramming may be used to generate therapeutically applicable iPSCs.

  • chromosomal transposition of piggybac in mouse embryonic stem cells
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Wei Wang, Dong Lu, Zeming Ning, David Melvin, Xiaozhong Wang, Allan Bradley
    Abstract:

    Transposon systems are widely used for generating mutations in various model organisms. PiggyBac (PB) has recently been shown to transpose efficiently in the mouse germ line and other mammalian cell lines. To facilitate PB's application in mammalian genetics, we characterized the properties of the PB Transposon in mouse embryonic stem (ES) cells. We first measured the transposition efficiencies of PB Transposon in mouse embryonic stem cells. We next constructed a PB/SB hybrid Transposon to compare PB and Sleeping Beauty (SB) Transposon systems and demonstrated that PB transposition was inhibited by DNA methylation. The excision and reintegration rates of a single PB from two independent genomic loci were measured and its ability to mutate genes with gene trap cassettes was tested. We examined PB's integration site distribution in the mouse genome and found that PB transposition exhibited local hopping. The comprehensive information from this study should facilitate further exploration of the potential of PB and SB DNA Transposons in mammalian genetics.

Edward V Mcassey - One of the best experts on this subject based on the ideXlab platform.

  • hybridization history and repetitive element content in the genome of a homoploid hybrid yucca gloriosa asparagaceae
    Frontiers in Plant Science, 2021
    Co-Authors: Karolina Heyduk, Edward V Mcassey, Jane Grimwood, Shengqiang Shu, Jeremy Schmutz, Michael R Mckain, Jim Leebensmack
    Abstract:

    Hybridization in plants results in phenotypic and genotypic perturbations that can have dramatic effects on hybrid physiology, ecology, and overall fitness. Hybridization can also perturb epigenetic control of transposable elements, resulting in their proliferation. Understanding the mechanisms that maintain genomic integrity after hybridization is often confounded by changes in ploidy that occur in hybrid plant species. Homoploid hybrid species, which have no change in chromosome number relative to their parents, offer an opportunity to study the genomic consequences of hybridization in the absence of change in ploidy. Yucca gloriosa (Asparagaceae) is a young homoploid hybrid species, resulting from a cross between Yucca aloifolia and Yucca filamentosa. Previous analyses of ~11kb of the chloroplast genome and nuclear-encoded microsatellites implicated a single Y. aloifolia genotype as the maternal parent of Y. gloriosa. Using whole genome resequencing, we assembled chloroplast genomes from 41 accessions of all three species to re-assess the hybrid origins of Y. gloriosa. We further used re-sequencing data to annotate Transposon abundance in the three species and mRNA-seq to analyze transcription of Transposons. The chloroplast phylogeny and haplotype analysis suggest multiple hybridization events contributing to the origin of Y. gloriosa, with both parental species acting as the maternal donor. Transposon abundance at the superfamily level was significantly different between the three species; the hybrid was frequently intermediate to the parental species in TE superfamily abundance or appeared more similar to one or the other parent. In only one case - Copia LTR Transposons - did Y. gloriosa have a significantly higher abundance relative to either parent. Expression patterns across the three species showed little increased transcriptional activity of Transposons, suggesting that either no Transposon release occurred in Y. gloriosa upon hybridization, or that any Transposons that were activated via hybridization were rapidly silenced. The identification and quantification of Transposon families paired with expression evidence paves the way for additional work seeking to link epigenetics with the important trait variation seen in this homoploid hybrid system.

  • hybridization history and repetitive element content in the genome of a homoploid hybrid yucca gloriosa asparagaceae
    bioRxiv, 2020
    Co-Authors: Karolina Heyduk, Edward V Mcassey, Jane Grimwood, Shengqiang Shu, Jeremy Schmutz, Michael R Mckain, Jim Leebensmack
    Abstract:

    Abstract Hybridization in plants results in phenotypic and genotypic perturbations that can have dramatic effects on hybrid physiology, ecology, and overall fitness. Hybridization can also perturb epigenetic control of transposable elements, resulting in their proliferation. Understanding the mechanisms that maintain genomic integrity after hybridization is often confounded by changes in ploidy that occur in hybrid plant species. Homoploid hybrid species, which have no change in chromosome number relative to their parents, offer an opportunity to study the genomic consequences of hybridization in the absence of change in ploidy. Yucca gloriosa (Asparagaceae) is a young homoploid hybrid species, resulting from a cross between Yucca aloifolia and Yucca filamentosa. Previous analyses of ~11kb of the chloroplast genome and nuclear-encoded microsatellites implicated a single Y. aloifolia genotype as the maternal parent of Y. gloriosa. Using whole genome resequencing, we assembled chloroplast genomes from multiple accessions of all three species to re-assess the hybrid origins of Y. gloriosa. We further used re-sequencing data to annotate Transposon abundance in the three species and mRNA-seq to analyze transcription of Transposons. The chloroplast phylogeny and haplotype analysis suggest multiple hybridization events contributing to the origin of Y. gloriosa, with both parental species acting as the maternal donor. Transposon abundance at the superfamily level was significantly different between the three species; the hybrid was frequently intermediate to the parental species in TE superfamily abundance or appeared more similar to one or the other parent. In only one case – Copia LTR Transposons – did Y. gloriosa have a significantly higher abundance relative to either parent. Expression patterns across the three species showed little increased transcriptional activity of Transposons, suggesting that either no Transposon release occurred in Y. gloriosa upon hybridization, or that any Transposons that were activated via hybridization were rapidly silenced. Further work will assess the degree to which Transposon abundance and location has affected the epigenomic landscape, gene expression, and ecophysiology in Y. gloriosa.