piggyBac Transposon-mediated Long-term Gene Expression in Mice

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

doi:10.1038/mt.2009.302

Cited by 82 publications

(80 citation statements)

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“…We and others have previously reported that piggyBac can mediate long-term gene expression in vivo following hydrodynamic tail-vein injection (Saridey et al, 2009;Nakanishi et al, 2010). Although SB100X has also been used for gene delivery by hydrodynamic injection (Mates et al, 2009), there has been no direct comparison of the various systems for in vivo gene delivery.…”

Section: Pb Increases Gene Delivery In Vivomentioning

confidence: 99%

“…PiggyBac has been shown to be capable of catalyzing transposition in a variety of organisms, including yeast (Mitra et al, 2008), Drosophila (Thibault et al, 2004), mouse somatic cells (Saridey et al, 2009;Nakanishi et al, 2010), mouse and human embryonic stem cells (Wang et al, 2008;Chen et al, 2010), and a variety of human somatic cell types (Wilson et al, 2007;Nakazawa et al, 2009;Woltjen et al, 2009). PiggyBac has a number of characteristics that make it flexible compared with other transposon systems, including its activity in a wide range of organisms, its ability to integrate multiple large transgenes with high efficiency (Kahlig et al, 2010), the ability to add domains to the transposase without loss of activity (Cadinanos and Bradley, 2007;Wilson et al, 2007), and excision from the genome without leaving a footprint mutation .…”

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Hyperactive piggyBac Gene Transfer in Human Cells and In Vivo

et al. 2012

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We characterized a recently developed hyperactive piggyBac (pB) transposase enzyme [containing seven mutations (7pB)] for gene transfer in human cells in vitro and to somatic cells in mice in vivo. Despite a protein level expression similar to that of native pB, 7pB significantly increased the gene transfer efficiency of a neomycin resistance cassette transposon in both HEK293 and HeLa cultured human cells. Native pB and SB100X, the most active transposase of the Sleeping Beauty transposon system, exhibited similar transposition efficiency in cultured human cell lines. When delivered to primary human T cells ex vivo, 7pB increased gene delivery two-to threefold compared with piggyBac and SB100X. The activity of hyperactive 7pB transposase was not affected by the addition of a 24-kDa N-terminal tag, whereas SB100X manifested a 50% reduction in transposition. Hyperactive 7pB was compared with native pB and SB100X in vivo in mice using hydrodynamic tail-vein injection of a limiting dose of transposase DNA combined with luciferase reporter transposons. We followed transgene expression for up to 6 months and observed approximately 10-fold greater long-term gene expression in mice injected with a codon-optimized version of 7pB compared with mice injected with native pB or SB100X. We conclude that hyperactive piggyBac elements can increase gene transfer in human cells and in vivo and should enable improved gene delivery using the piggyBac transposon system in a variety of cell and gene-therapy applications.

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Section: Pb Increases Gene Delivery In Vivomentioning

confidence: 99%

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confidence: 99%

Hyperactive piggyBac Gene Transfer in Human Cells and In Vivo

et al. 2012

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“…To evaluate the role of IL-4 in vivo, we expressed IL-4 in mice by injecting a plasmid based on a transposon expression system using hydrodynamic injection (29)(30)(31). In this system, the gene of interest integrates into the genome in a site-specific fashion, primarily in hepatocytes, allowing for efficient and high-level expression of the introduced gene (32).…”

Section: Il-4 Overexpression Induces Proteinuria and Il-4 Signaling Imentioning

confidence: 99%

B cell–derived IL-4 acts on podocytes to induce proteinuria and foot process effacement

et al. 2017

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“…4,23) Using this Gluc expressing pDNA, we examined the effect of different doses of the donor and helper pDNAs on the sustained gene expression in mouse liver after hydrodynamic injection. In our research, mice were co-transfected with a constant amount (25 µg) of pORF-Gluc/attB that induced saturated expression of Gluc and a different amount (1, 10, 25, 50 µg) of the helper pDNA (pCMV-int, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…[1][2][3] Transposons have been used as an effective method of genomic integration in vivo, but they also integrate randomly. 4) Therefore, the use of site-specific recombinases such as Cre and Flp could be a promising method; however, their main utility is in creating deletions mediated by the reversibility of their enzyme reactions. 5,6) It was reported that bacteriophage phiC31 integrase performs a site-specific and unidirectional reaction.…”

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Evaluation of Long-Term Gene Expression in Mouse Liver Using PhiC31 Integrase and Hydrodynamic Injection

Umemoto

Kawakami

Otani

et al. 2012

Biological & Pharmaceutical Bulletin

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PhiC31 integrase has the potential to achieve long-term transgene expression because of site-specific and unidirectional recombination. In this study, plasmid DNA (pDNA) encoding Gaussia luciferase (Gluc) cDNA was constructed and the optimal condition for long-term gene expression using phiC31 integrase in mouse liver after hydrodynamic injection was investigated. Gluc is secreted and thus allows quantification of its expression level in blood and easy determination of the expression time-course. Mice were co-transfected with 25 µg donor pDNA (pORF-Gluc/attB) and different amounts of helper pDNA (pCMV-int; from 1 to 50 µg). Serum Gluc expression was evaluated during 120 d. The most highly sustained gene expression was obtained when 10 µg of helper pDNA was co-transfected in ICR and C57BL/6 mice. However, the Gluc expression in C57BL/6 mice was slightly lower and less durable than that in ICR mice. Little hepatic damage caused by phiC31 integrase was observed during 120 d in ICR and C57BL/6 mice.Key words non-viral gene delivery; gene vector; plasmid DNA; hepatocyte A variety of strategies for long-term gene expression have been developed. However, many obstacles that hinder safe and effective gene therapy remain. For example, retroviral vectors, which induce highly efficient integration, mediate random integration into genomic DNA that activated oncogenes both in animals and in clinical trials.1-3) Transposons have been used as an effective method of genomic integration in vivo, but they also integrate randomly.4) Therefore, the use of site-specific recombinases such as Cre and Flp could be a promising method; however, their main utility is in creating deletions mediated by the reversibility of their enzyme reactions. 5,6) It was reported that bacteriophage phiC31 integrase performs a site-specific and unidirectional reaction. 7) In nature, phiC31 integrase mediates the recombination of the phage genome into the bacterial chromosome via a reaction between the phage attP site and the bacterial attB site. 8) Recently, Thyagarajan et al. have demonstrated that phiC31 integrase can act in cultured mammalian cells and that it can efficiently recombine plasmid DNA (pDNA) that includes an attB site into so called pseudo attP sites in mammalian genomic DNA.9) In addition, the number of potential sites integrated by phiC31 integrase in the human genome was estimated at ca. 370 sites, and the sites appear to be in comparatively safe locations. 10)So far, the use of phiC31 integrase for prolonged gene expression has been demonstrated in mice [11][12][13] and in cultured cells. 9,14) This system requires the use of two types of pDNAs: the donor pDNA encoding attB and the helper pDNA encoding phiC31 integrase. Hydrodynamic injection is a transfection method that permits high gene expression in the liver and there are many reports combining this method with phiC31 integrase for basic research using firefly luciferase, genes related to genetic diseases or hepatic murine cancer model. 11,[15][16][17][18][19][20][21] Taking thes...

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piggyBac Transposon-mediated Long-term Gene Expression in Mice

Cited by 82 publications

References 47 publications

Hyperactive piggyBac Gene Transfer in Human Cells and In Vivo

Hyperactive piggyBac Gene Transfer in Human Cells and In Vivo

B cell–derived IL-4 acts on podocytes to induce proteinuria and foot process effacement

Evaluation of Long-Term Gene Expression in Mouse Liver Using PhiC31 Integrase and Hydrodynamic Injection

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