Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase

Elick, Teresa A.; Bauser, Christopher A.; Fraser, Malcolm J.

doi:10.1007/bf00120216

Cited by 129 publications

(104 citation statements)

References 37 publications

(38 reference statements)

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“…It is active in many cell types, including mammalian cells (9,14,25), and is distinguished by its ability to excise precisely (11), restoring the donor site to its preinsertion sequence. In addition, PB and iPB7 remain active when fused to heterologous DNA-binding domains.…”

Section: Discussionmentioning

confidence: 99%

“…piggyBac, originally isolated from the cabbage looper moth Trichoplusia ni genome (3), has a large cargo size (4), is highly active in many cell types, and mediates long-term expression in mammalian cells in vivo (5)(6)(7)(8)(9)(10). piggyBac is also distinguished by its ability to excise precisely (11), thus restoring the donor site to its pretransposon insertion sequence.…”

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confidence: 99%

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piggyBac transposase tools for genome engineering

Burnight

Cooney

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

209

193

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The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc + Int − ) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc + Int − transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int − phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc + Int − transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction.NA "cut-and-paste" transposable elements are important tools for genome engineering, such as insertional mutagenesis and transgenesis. Research with the DNA transposon Sleeping Beauty, a "resurrected" transposon, has pioneered the use of DNA transposons in mammalian cells (1, 2). piggyBac is also a DNA transposon and a promising alternative to Sleeping Beauty. piggyBac, originally isolated from the cabbage looper moth Trichoplusia ni genome (3), has a large cargo size (4), is highly active in many cell types, and mediates long-term expression in mammalian cells in vivo (5-10). piggyBac is also distinguished by its ability to excise precisely (11), thus restoring the donor site to its pretransposon insertion sequence.Because it can excise precisely, piggyBac is especially useful if a transgene is only transiently required. Transient integration and expression of transcription factors are important approaches to generate transgene-free induced pluripotent stem cells (iPSCs) (12, 13) as well as directed differentiation of specific cell types for both research and clinical use. Removal of the transgenes is key for potential therapeutic applications of iPSCs. piggyBac has been used as a vector for reversible integration; however, reintegration of the transposon catalyzed by piggyBac (PB) transposase occurs in 40-50% of cells (14) Int− transposase whose excision frequency is five-to six-f...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

piggyBac transposase tools for genome engineering

Burnight

Cooney

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

209

193

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“…On the other hand, the precise excision mechanism may harbor advantages as well. piggyBac transposons leave no target site duplication (footprint) when they are excised (45) and, therefore, allow restoration of insertion loci to wild type even when the transposon is inserted in the ORF. In contrast to P elements, piggyBac is therefore less likely to cause secondary mutations by a hit-and-run mechanism, which can severely complicate the identification of mutations.…”

Section: Discussionmentioning

confidence: 99%

piggyBac -based insertional mutagenesis in the presence of stably integrated P elements in Drosophila

Häcker

Nystedt

Barmchi

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS͞Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jumpstarter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms. A t a time when whole genome sequences for the most important developmental biology model organisms have become available, the focus of developmental biologists is turning toward the functional analysis of whole genomes. The most straightforward strategy for the identification of gene functions relies on the inactivation of individual transcription units, by either random or site-directed mutagenesis. In Drosophila, an estimated 25% of all vital genes have been mutated to date (1, 2). Mutagenesis efforts have been based largely on chemically induced random mutagenesis by using ethane methyl sulfonate (3) or on transposon-mediated insertional mutagenesis by using P elements (4, 5). Unfortunately, both of these methods have limitations, which make the mutation of all genes in the Drosophila genome difficult to attain.Mapping of chemically induced mutations at the DNA level is labor intensive and time consuming and therefore not practical on a genome-wide basis. In contrast, P element-induced mutations can easily and quickly be identified on a large scale. However, the tendency of P elements to integrate preferentially into certain hotspots (1) makes saturation mutagenesis difficult, if not impossible. Moreover, genetic screens based on the isolation of zygotic lethal mutations allow only the detection of the earliest function of a given gene. The analysis of later gen...

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“…In contrast, piggyBac class II transposons generate no footprint at their donor site, which is reconstituted precisely upon excision to restore the initial 59-TTAA-39 target sequence (Elick et al 1996). In vitro, the piggyBac transposase catalyzes double-strand cleavage at transposon ends, and the resulting DSBs exhibit 4-base 59 overhangs carrying the duplicated TTAA target site (Mitra et al 2008).…”

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confidence: 99%

PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia

Baudry¹,

Malinsky²,

Restituito³

et al. 2009

Genes Dev.

185

326

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Programmed genome rearrangements drive functional gene assembly in ciliates during the development of the somatic macronucleus. The elimination of germline sequences is directed by noncoding RNAs and is initiated by DNA double-strand breaks, but the enzymes responsible for DNA cleavage have not been identified. We show here that PiggyMac (Pgm), a domesticated piggyBac transposase, is required for these rearrangements in Paramecium tetraurelia. A GFP-Pgm fusion localizes in developing macronuclei, where rearrangements take place, and RNAi-mediated silencing of PGM abolishes DNA cleavage. This is the first in vivo evidence suggesting an essential endonucleolytic function of a domesticated piggyBac transposase.Supplemental material is available at http://www.genesdev.org.

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Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase

Cited by 129 publications

References 37 publications

piggyBac transposase tools for genome engineering

piggyBac transposase tools for genome engineering

piggyBac -based insertional mutagenesis in the presence of stably integrated P elements in Drosophila

PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia

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