Comparison of piggyBac transposition efficiency between linear and circular donor vectors in mammalian cells

Nakanishi, Hiizu; Higuchi, Yuriko; Kawakami, Shigeru; Yamashita, Fumiyoshi; Hashida, Mitsuru

doi:10.1016/j.jbiotec.2011.05.009

Cited by 11 publications

(11 citation statements)

References 20 publications

(26 reference statements)

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“…Therefore, we first determined optimal transfection conditions for our model CHO cells. Different quantities and ratios of plasmid bearing the puromycin resistance-carrying transposon and of the PB transposase expression vector were co-transfected into cultured cells, and the occurrence of antibiotic-resistant colonies were scored (Figure S1A and S1B), as performed in earlier studies [17], [18]. The frequency of puromycin-resistant colonies was increased up to 5-fold in the presence of the transposase, when compared to spontaneous genomic integration of the plasmids upon co-transfections with a PB-devoid control plasmid (Figure S1C and data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…Cells were transfected with various amounts of a supercoiled plasmid encoding the PB transposase (ranging from 0 to 1500 ng) for titration experiments or co-transfected with the optimal ratio of 300 ng of PB transposase expression plasmid and 300 ng of transposon donor plasmid. Supercoiled transposon-donor plasmids were used, as it was shown that PB transposition from linear vector is relatively inefficient when compared to circular transposon plasmids [18]. Plasmids transfected as controls without the transposase were also supercoiled, as the use of linearized plasmids did not increase significantly the frequency of occurrence of spontaneous integration ( < 2-fold, data not shown).…”

Section: Methodsmentioning

confidence: 99%

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MAR Elements and Transposons for Improved Transgene Integration and Expression

Ley

Harraghy

Fourn³

et al. 2013

PLoS ONE

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Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1–68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1–68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2–4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

MAR Elements and Transposons for Improved Transgene Integration and Expression

Ley

Harraghy

Fourn³

et al. 2013

PLoS ONE

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“…Minimal piggyBac vectors have equal integration/transposition efficiency regardless of whether the delivered plasmid was open or circular whereas in classical, full-length transposons transposition efficiency is higher when delivered as circular DNA. 28 This creates the possibility of combining the minimal piggyBac vector with other strategies such as the use of integration-deficient retroviruses for DNA delivery. 29 …”

Section: Discussionmentioning

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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“…Therefore, we first determined optimal transfection conditions for our model CHO cells. Different quantities and ratios of plasmid bearing the puromycin resistance-carrying transposon and of the PB transposase expression vector were co-transfected into cultured cells, and the occurrence of antibiotic-resistant colonies were scored ( Figure S1A and S1B), as performed in earlier studies [17,18]. The frequency of puromycin-resistant colonies was increased up to 5-fold in the presence of the transposase, when compared to spontaneous genomic integration of the plasmids upon cotransfections with a PB-devoid control plasmid ( Figure S1C and data not shown).…”

Section: Effect Of Mar Inclusion On Transposition Efficiencymentioning

confidence: 99%

Section: Cell Culture and Transfection Analysismentioning

confidence: 99%