Minimal piggyBac vectors for chromatin integration

Solodushko, Viktoriya; Bitko, Vira; Fouty, Brian

doi:10.1038/gt.2013.52

Cited by 18 publications

(35 citation statements)

References 21 publications

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“…Nonetheless, our findings of a high rate of transposase integration when using a ‘cis’ configuration are important as the use of PB for genome modification increases and ‘cis’ plasmid vectors are available. A recent investigation reported integration of ‘minimal’ PB components when full-length TRs are provided elsewhere in the plasmid; however, these results can be explained via our observation of transposon plasmid backbone DNA integration ( 33 ).…”

Section: Discussionmentioning

confidence: 68%

“…Therefore, we observed no mobilization of endogenous human genomic DNA by the PB transposase using colony count assays, excision assays and double-stranded DNA break assays. Further refinement of PB vectors has demonstrated that shortening the 5′TR to 35 bp and the 3′TR to 63 bp may still mediate gene transfer ( 33 ). It is likely that a high degree of sequence specificity is required to mediate transposition from both TRs.…”

Section: Resultsmentioning

confidence: 99%

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Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

Saha

Woodard

Charron

et al. 2015

Nucleic Acids Research

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Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.

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Section: Discussionmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 99%

Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

Saha

Woodard

Charron

et al. 2015

Nucleic Acids Research

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“…In previous studies, we detailed its construction and demonstrated its ability to transpose cells as efficiently as full-length piggyBac transposons 11 ; we also demonstrated that this vector can be used for the rapid generation and purification of packaging cells capable of stably producing self-inactivated gamma-retroviruses. 26 Here, we show that this vector can be used to successfully integrate a 15 kb transgenic sequence into target cells, twice the capacity of retroviral vectors which are currently the most popular method for stable gene delivery.…”

Section: Discussionmentioning

confidence: 99%

“…10 Recently, we developed a modified piggyBac delivery system in which most of both terminal domains were relocated from the delivery cassette into the helper (nonintegrating) part of the same plasmid to minimize the size of the delivered transposon; this was accomplished without a significant loss of transposition efficiency. 11 Despite the reduction in the size of the delivered fragment, these minimal piggyBac plasmids include all the required elements for transposon integration. Like classical piggyBac plasmids, these minimal piggyBac vectors have two segments—one segment that is integrated and one that facilitates this integration.…”

Section: Introductionmentioning

confidence: 99%

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

Troyanovsky

Bitko

Pastukh

et al. 2016

Molecular Therapy - Nucleic Acids

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Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase) when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon) vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.

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“…Recently we developed a modified piggyBac transposon delivery system in which the transposase gene and most of both terminal domains were relocated from the delivery cassette into the helper (non-integrating) part of the same plasmid without a significant loss of transposition efficiency [ 17 ]. Similar to classical piggyBac vectors, these truncated (minimal) transposon vectors can deliver a large amount of transgenic DNA, yet incorporate significantly less helper DNA (fewer than 100 base pairs) than classical piggyBac vectors or retroviruses.…”

Section: Introductionmentioning

confidence: 99%

Simple viral/minimal piggyBac hybrid vectors for stable production of self-inactivating gamma-retroviruses

et al. 2015

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Background Transient production of gamma-retroviruses, including self-inactivating (SIN) retroviruses, is a common method for rapidly generating virus capable of gene delivery. Stable (continuous) production of virus is preferable to transient production for clinical and biotechnology purposes, however, because it allows for significant quantities of a uniform virus to be generated over a prolonged period of time, thus allowing for longitudinal functional studies and quality analysis. Unfortunately, stable production of SIN retroviruses is difficult to achieve. ResultsWe describe a novel method to rapidly and cost-effectively create packaging cells capable of continuously producing self-inactivating gamma-retroviruses. We imbedded the SIN proviral construct into a minimal piggyBac transposon vector and then integrated the hybrid vector into packaging cells that already stably expressed the viral gag-pro-pol and envelope genes. Cells that stably produced self-inactivating gamma-retroviruses could be identified (and purified) as early as 3 weeks after initial transfection; these cells produced virus for at least 9 weeks without a decline in titer.ConclusionsThis viral-minimal piggyBac hybrid vector allowed for the rapid generation and purification of packaging cells capable of stably producing self-inactivated gamma-retroviruses. This method can be applied to the large-scale production of viruses for use in research, biotechnology, and potentially, clinical trials.

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Minimal piggyBac vectors for chromatin integration

Cited by 18 publications

References 21 publications

Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

Simple viral/minimal piggyBac hybrid vectors for stable production of self-inactivating gamma-retroviruses

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