RNA as a Source of Transposase for Sleeping Beauty-Mediated Gene Insertion and Expression in Somatic Cells and Tissues

Wilber, Andrew; Frandsen, Joel L.; Geurts, Jennifer L.; Largaespada, David A.; Hackett, Perry B.; McIvor, R. Scott

doi:10.1016/j.ymthe.2005.10.014

Cited by 107 publications

(89 citation statements)

References 32 publications

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“…Instability of mRNA restricts its duration of activity and therefore the risk of introducing genotoxic effects. However, it is possible for the mRNA to undergo reverse transcription, potentially resulting in insertion of the pBt cDNA into the host genome by nonhomologous recombination (31). We modified our helper-independent piGENIE plasmid in two steps, thereby significantly improving its safety.…”

Section: Discussionmentioning

confidence: 99%

Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

Urschitz

Kawasumi

Owens

et al. 2010

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

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Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

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Section: Discussionmentioning

confidence: 99%

Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

Urschitz

Kawasumi

Owens

et al. 2010

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

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“…This is an important finding since continued expression of the transposase could potentially facilitate transposon re-mobilization and re-insertion, resulting in reduced overall stability of the pig model. Although this aspect is rarely experimentally addressed in reports describing transposon-directed gene delivery, methods for co-delivery of transposon donor plasmid with in vitro-synthesized mRNA encoding the transposase to somatic cells have been described (Wilber et al 2006) and may be applicable in protocols of SB-directed pig transgenesis. Also, the use of in vitro-transcribed mRNA as a source of transposase has been successful in early embryos of frog (Sinzelle et al 2006), zebrafish (Davidson et al 2003), and mouse (Dupuy et al 2002).…”

Section: Discussionmentioning

confidence: 99%

Pig transgenesis by Sleeping Beauty DNA transposition

Jakobsen

Kragh

et al. 2010

Transgenic Res

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Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.

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“…Also shown are the percentages of successfully transfected animals which are born (Ab) using various delivery methods. Transfection rates with PgB:ICSI are comparable to Lentiviral Vectors in both percentage of oocytes transfected and animals born source for the transposase enzymatic activity, which has been shown to be effective (Wilber et al 2006). The transient nature of cRNA, limits the duration of transposase activity and would likely attenuate the risks of the integration of the transposase into the host genome.…”

Section: Active Transgenesismentioning

confidence: 99%

Active integration: new strategies for transgenesis

Shinohara

Kaminski²,

Segal

et al. 2007

Transgenic Res

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This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

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RNA as a Source of Transposase for Sleeping Beauty-Mediated Gene Insertion and Expression in Somatic Cells and Tissues

Cited by 107 publications

References 32 publications

Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

Pig transgenesis by Sleeping Beauty DNA transposition

Active integration: new strategies for transgenesis

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