The Experts below are selected from a list of 19083 Experts worldwide ranked by ideXlab platform
Zsuzsanna Izsvák - One of the best experts on this subject based on the ideXlab platform.
-
suicidal autointegration of sleeping beauty and piggybac Transposons in eukaryotic cells
PLOS Genetics, 2014Co-Authors: Yongming Wang, Zoltán Ivics, Jichang Wang, Anatharam Devaraj, Manvendra Singh, Ana Jimenez Orgaz, Jiaxuan Chen, Matthias Selbach, Zsuzsanna IzsvákAbstract:Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. ‘Cut and paste’ DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA Transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these Transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naive cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised Transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger Transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both Transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.
-
Transposon-mediated genome manipulation in vertebrates
Nature Methods, 2009Co-Authors: Zoltán Ivics, Allan Bradley, Jef D Boeke, Lajos Mátés, Andras Nagy, Zsuzsanna IzsvákAbstract: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, 2000Co-Authors: Zoltán Ivics, Zsuzsanna Izsvák, Ronald H A PlasterkAbstract: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, 1997Co-Authors: Zoltán Ivics, Zsuzsanna Izsvák, Perry B Hackett, Ronald H A PlasterkAbstract: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.
Ruth M. Hall - One of the best experts on this subject based on the ideXlab platform.
-
is26 mediated formation of Transposons carrying antibiotic resistance genes
mSphere, 2016Co-Authors: Christopher J Harmer, Ruth M. HallAbstract:ABSTRACT The IS26 transposase, Tnp26, catalyzes IS26 movement to a new site and deletion or inversion of adjacent DNA via a replicative route. The intramolecular deletion reaction produces a circular molecule consisting of a DNA segment and a single IS26, which we call a translocatable unit or TU. Recently, Tnp26 was shown to catalyze an additional intermolecular, conservative reaction between two preexisting copies of IS26 in different plasmids. Here, we have investigated the relative contributions of homologous recombination and Tnp26-catalyzed reactions to the generation of a transposon from a TU. Circular TUs containing the aphA1a kanamycin and neomycin resistance gene or the tet(D) tetracycline resistance determinant were generated in vitro and transformed into Escherichia coli recA cells carrying R388::IS26. The TU incorporated next to the IS26 in R388::IS26 forms a transposon with the insertion sequence (IS) in direct orientation. Introduction of a second TU produced regions containing both the aphA1a gene and the tet(D) determinant in either order but with only three copies of IS26. The integration reaction, which required a preexisting IS26, was precise and conservative and was 50-fold more efficient when both IS26 copies could produce an active Tnp26. When both ISs were inactivated by a frameshift in tnp26, TU incorporation was not detected in E. coli recA cells, but it did occur in E. coli recA+ cells. However, the Tnp-catalyzed reaction was 100-fold more efficient than RecA-dependent homologous recombination. The ability of Tnp26 to function in either a replicative or conservative mode is likely to explain the prominence of IS26-bounded Transposons in the resistance regions found in Gram-negative bacteria. IMPORTANCE In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming Transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel conservative movement mechanism in which an incoming IS26 targets a preexisting one. Here, we have demonstrated how IS26-bounded class I Transposons can be produced from translocatable units (TUs) containing only an IS26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS26, creating a class I transposon, and if the targeted IS26 is in a transposon, the product resembles two Transposons sharing a central IS26, a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS26 builds Transposons and brings additional Transposons into resistance regions.
-
Distribution of the blaTEM gene and blaTEM-containing Transposons in commensal Escherichia coli
The Journal of antimicrobial chemotherapy, 2011Co-Authors: Jannine Bailey, Jeremy L. Pinyon, Sashindran Anantham, Ruth M. HallAbstract:Objectives: The context of antibiotic resistance genes can provide valuable information about the epidemiology of mobile genetic elements. This study examined the distribution of the closely related bla TEM Transposons Tn1, Tn2 and Tn3, or bla TEM -containing fragments of them, in ampicillin-resistant human commensal Escherichia coli isolates. Methods: A PCR mapping protocol was used to detect different segments of the Transposons or to link partial copies to the insertion sequence IS26. Restriction digestion of one amplicon was used to assign Transposons to Tn1, Tn2 or Tn3 groups and sequencing validated this approach. Restriction digestion and sequencing were used to determine how much of the transposon remained when bla TEM was linked to IS26. Sequences were compared with those in GenBank. Results: Of 25 ampicillin-resistant E. coli strains recovered from the faecal flora of healthy humans that carried the bla TEM gene, 15 carried a complete copy of Tn2 or a Tn2 variant; one was interrupted by IS4. A further isolate carried Tn3. Tn2 was also most abundant in sequences available in GenBank. Two isolates carried Tn2 and an IS26-bla TEM fragment. The remaining 10 isolates carried only the bla TEM end of the transposon and 9 of these partial copies were flanked by IS26 at varying distances upstream of bla TEM . One configuration corresponded to that in Tn6029B and the complete transposon was shown to be present. Conclusions: Tn1, Tn2 and Tn3 can be simply and rapidly identified. Tn2 appears to be the most widely distributed. However, the bla TEM -containing end associated with an IS26 is also widely distributed.
-
Transposons related to tn1696 in inchi2 plasmids in multiply antibiotic resistant salmonella enterica serovar typhimurium from australian animals
Microbial Drug Resistance, 2010Co-Authors: Amy K Cain, Xiulan Liu, Steven P Djordjevic, Ruth M. HallAbstract:Conjugative IncHI2 plasmids carrying tetracycline, trimethoprim, and sulphonamide resistance genes were recovered from two multiply antibiotic resistant Salmonella enterica serovar Typhimurium isolates from Australian food-producing animals. Transposons related to the mercury resistance transposon Tn1696 were identified in both IncHI2 plasmids. These Transposons contained an In4-type class 1 integron that carried a dfrA5 trimethoprim resistance gene cassette and the sul1 sulfonamide resistance gene. These integrons were located in the same position as In4 in Tn1696. The integron from one isolate includes a large transposon-like structure containing four IS26 and the strAB, sul2, blaTEM, and aphA1 genes conferring resistance to streptomycin, sulphonamides, ampicillin, kanamycin, and neomycin, respectively. This structure is flanked by an 8-bp duplication, but it includes both the aphA1-containing transposon Tn4352 and a transposon, Tn6029, carrying genes derived from RSF1010 and from Tn2. However, Tn4352 a...
-
Transposons tn1696 and tn21 and their integrons in4 and in2 have independent origins
Antimicrobial Agents and Chemotherapy, 2001Co-Authors: Sally R Partridge, H. W. Stokes, Heidi J Brown, Ruth M. HallAbstract:The first 13.6 kb of the mercury and multidrug resistance transposon Tn1696, which includes the class 1 integron In4, has been sequenced. In4 is 8.33 kb long and contains the 5′-conserved segment (5′-CS) and 2.24 kb of the 3′-conserved segment (3′-CS) flanking four integrated cassettes. The 3′-CS region is followed by one full copy and an adjacent partial copy of the insertion sequence IS6100 flanked, in inverse orientation, by two short segments (123 and 152 bp) from the outer right-hand end of class 1 integrons. This structure is representative of a distinct group of class 1 integrons that differs from In2, found in Tn21, and other related class 1 integrons. In4 does not include transposition genes but is bounded by characteristic 25-bp inverted repeats and flanked by a direct duplication of 5 bp of the target sequence, indicating that it was inserted by a transpositional mechanism. In4 lies between the resII and resI sites of a backbone mercury resistance transposon which is >99.5% identical to Tn5036. Although Tn21 and Tn1696 are both classified as members of the Tn21 subfamily of the Tn3 transposon family, the backbone mercury resistance Transposons are only 79 to 96% identical. Tn21 also contains a region of about 0.7 kb not found in Tn1696. The integrons In2 and In4 carrying the antibiotic resistance genes have been inserted at different locations into distinct ancestral mercury resistance Transposons. Thus, Tn21 and Tn1696 have independent histories and origins. Other Transposons (Tn1403 and Tn1412) that include a class 1 integron also have independent origins. In all except Tn21, the integron is located within the res region of the backbone transposon.
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, 2021Co-Authors: Karolina Heyduk, Edward V Mcassey, Jane Grimwood, Shengqiang Shu, Jeremy Schmutz, Michael R Mckain, Jim LeebensmackAbstract: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, 2020Co-Authors: Karolina Heyduk, Edward V Mcassey, Jane Grimwood, Shengqiang Shu, Jeremy Schmutz, Michael R Mckain, Jim LeebensmackAbstract: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.
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, 2021Co-Authors: Karolina Heyduk, Edward V Mcassey, Jane Grimwood, Shengqiang Shu, Jeremy Schmutz, Michael R Mckain, Jim LeebensmackAbstract: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, 2020Co-Authors: Karolina Heyduk, Edward V Mcassey, Jane Grimwood, Shengqiang Shu, Jeremy Schmutz, Michael R Mckain, Jim LeebensmackAbstract: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.
-
PiggyBac Transposon-Based Insertional Mutagenesis in Mice.
Methods in Molecular Biology, 2018Co-Authors: Mathias Friedrich, Iraad F. Bronner, Pentao Liu, Allan Bradley, Roland RadAbstract:While sequencing and array-based studies are creating catalogues of genetic alterations in cancer, discriminating cancer drivers among the large sets of epigenetically, transcriptionally or posttranslationally dysregulated genes remains a challenge. Transposon-based genetic screening in mice has proven to be a powerful approach to address this challenge. Insertional mutagenesis directly flags biologically relevant genes and, combined with the transposon's unique molecular fingerprint, facilitates the recovery of insertion sites. We have generated transgenic mouse lines harboring different versions of PiggyBac-based oncogenic Transposons, which in conjunction with PiggyBac transposase mice can be used for whole-body or tissue-specific insertional mutagenesis screens. We have also developed QiSeq, a method for (semi-)quantitative transposon insertion site sequencing, which overcomes biasing limitations of previous library preparation methods. QiSeq can be used in multiplexed high-throughput formats for candidate cancer gene discovery and gives insights into the clonal distribution of insertions for the study of genetic tumor evolution.
-
mobilization of giant piggybac Transposons in the mouse genome
Nucleic Acids Research, 2011Co-Authors: Daniel J Turner, Nancy L Craig, Kosuke Yusa, Zemin Ning, Qi Liang, Sabine E Eckert, Lena Rad, Tomas W Fitzgerald, Allan BradleyAbstract:The development of technologies that allow the stable delivery of large genomic DNA fragments in mammalian systems is important for genetic studies as well as for applications in gene therapy. DNA Transposons have emerged as flexible and efficient molecular vehicles to mediate stable cargo transfer. However, the ability to carry DNA fragments >10 kb is limited in most DNA Transposons. Here, we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant Transposons express the genomic cargo. Furthermore, these 100-kb Transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications.
-
Transposon-mediated genome manipulation in vertebrates
Nature Methods, 2009Co-Authors: Zoltán Ivics, Allan Bradley, Jef D Boeke, Lajos Mátés, Andras Nagy, Zsuzsanna IzsvákAbstract: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.
-
chromosomal mobilization and reintegration of sleeping beauty and piggybac Transposons
Genesis, 2009Co-Authors: Qi Liang, Jun Kong, James Stalker, Allan BradleyAbstract:The Sleeping Beauty and PiggyBac DNA transposon systems have recently been developed as tools for insertional mutagenesis. We have compared the chromosomal mobilization efficiency and insertion site preference of the two Transposons mobilized from the same donor site in mouse embryonic stem (ES) cells under conditions in which there were no selective constraints on the Transposons' insertion sites. Compared with Sleeping Beauty, PiggyBac exhibits higher transposition efficiencies, no evidence for local hopping and a significant bias toward reintegration in intragenic regions, which demonstrate its utility for insertional mutagenesis. Although Sleeping Beauty had no detectable genomic bias with respect to insertions in genes or intergenic regions, both Sleeping Beauty and PiggyBac Transposons displayed preferential integration into actively transcribed loci. genesis 47:404–408, 2009. © 2009 Wiley-Liss, Inc.
-
generation of transgene free induced pluripotent mouse stem cells by the piggybac transposon
Nature Methods, 2009Co-Authors: Kosuke Yusa, Roland Rad, Junji Takeda, Allan BradleyAbstract: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.
