Hyperactive<i>Himar1</i>Transposase Mediates Transposition in Cell Culture and Enhances Gene Expression<i>In Vivo</i>

Keravala, Annahita; Liu, Dexi; Lechman, Eric R.; Wolfe, Darren; Nash, Joan; Lampe, David J.; Robbins, Paul D.

doi:10.1089/hum.2006.17.1006

Cited by 29 publications

(22 citation statements)

References 36 publications

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“…This element has been shown to be active in a number of animals, including mammals [52,53]. Hyperactive variants have been developed as well [54]. How this element quantitatively compares to other members of this family in vertebrate applications is still largely unknown.…”

Section: Himar1mentioning

confidence: 99%

Transposon tools hopping in vertebrates

Ni¹,

Clark²,

Fahrenkrug³

et al. 2008

Briefings in Functional Genomics and Proteomics

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In the past decade, tools derived from DNA transposons have made major contributions to vertebrate genetic studies from gene delivery to gene discovery. Multiple, highly complementary systems have been developed, and many more are in the pipeline. Judging which DNA transposon element will work the best in diverse uses from zebrafish genetic manipulation to human gene therapy is currently a complex task. We have summarized the major transposon vector systems active in vertebrates, comparing and contrasting known critical biochemical and in vivo properties, for future tool design and new genetic applications.

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Section: Himar1mentioning

confidence: 99%

Transposon tools hopping in vertebrates

Ni¹,

Clark²,

Fahrenkrug³

et al. 2008

Briefings in Functional Genomics and Proteomics

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“…The Sleeping Beauty transposon system represents an increasingly important vehicle for in vivo gene delivery. At present, however, much of the effort directed towards successfully adapting this and other DNA transposons to a clinical setting have focused to a large extent on obtaining improved integration frequencies in target cells via mutation of transposon (donor) and/or transposase (helper) components (1,13,27,60,64). Our work presented herein suggests that SB's integration efficiency may not be as great a barrier as previously thought and implies that a greater emphasis on postintegrative regulatory mechanisms may ultimately prove more productive in perfecting SB for a clinical environment.…”

Section: Vol 27 2007 Transposon Silencing In Mammals 8829mentioning

confidence: 99%

Postintegrative Gene Silencing within the Sleeping Beauty Transposition System

Garrison

Yant

Mikkelsen

et al. 2007

Molecular and Cellular Biology

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The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can efficiently and stably integrate into mammalian genomes. In this report, we examined transposon expression in human cells using a novel nonselective fluorescence-activated cell sorter-based method and discovered that SB integrates ϳ20 times more frequently than previously reported within systems that were dependent on transgene expression and likely subject to postintegrative gene silencing. Over time, phenotypic analysis of clonal integrants demonstrated that SB undergoes additional postintegrative gene silencing, which varied based on the promoter used for transgene expression. Molecular and biochemical studies suggested that transposon silencing was influenced by DNA methylation and histone deacetylation because both 5-aza-2-deoxycytidine and trichostatin A partially rescued transgene silencing in clonal cell lines. Collectively, these data reveal the existence of a multicomponent postintegrative gene silencing network that efficiently targets invading transposon sequences for transcriptional silencing in mammalian cells.Transposable elements play an important role in evolution which is demonstrated by an estimated 50 genes within the human genome that are transposon derived, including genes vital to our adaptive immunity (16) and DNA repair (33). Amazingly, sequences recognized as derived from transposable elements account for approximately 45% of the overall human genome (31). Transposable elements can be broken down into two main groups: those which rely on an RNA intermediate for remobilization (retro-elements) and those which are remobilized directly as DNA (DNA transposons). Fossils of one group of DNA transposons, the Tc1/mariner type, are found throughout nature. Remnants, which have been deactivated through the accumulation of mutations over time (21,22), have been isolated from a variety of organisms, including fish (22), Xenopus laevis (30, 51), insects (49), Caenorhabditis elegans (48), Drosophila melanogaster (47), and humans (43, 52). Recognizing the many potential uses of an active and efficient DNA transposon system, Ivics et al. reconstructed a functional Tc1/ mariner-like element from inactive transposon remnants found in fish and named it Sleeping Beauty (SB) (21). Shortly following its description, our group established the possible utility of this system as a means to genetically modify somatic tissues in adult mammals to treat animal models of human disease (40,57,59).DNA transposons of the Tc1/mariner type contain a simple structure in which the only components required for transposition are inverted repeats (IRs), which flank the DNA to be transposed, and Sleeping Beauty transposase. As with other DNA transposons, SB transposition occurs through a "cut and paste" mechanism mediated by binding to the IRs of Sleeping Beauty transposase, which can be supplied either in cis from an autonomous element or in trans from a nonautonomous element. The excision and integration steps are me...

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“…There is currently work being done with PB transposase to increase the transpositional efficiencies to that of retroviral vectors. There are methods to accomplish this goal such as PCR random mutagenesis or alanine substitutions utilizing mutagenic PCR primers (Goryshin and Reznikoff 1998;Yant et al 2004;Pledger and Coates 2005;Keravala et al 2006). One of us, Thomas Ryan, is pursuing PB active transgenesis in embryonic stem cells for the production of transgenic animals.…”

Section: Mechanisms To Improve Specificity and Efficiency Of Transfecmentioning

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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HyperactiveHimar1Transposase Mediates Transposition in Cell Culture and Enhances Gene ExpressionIn Vivo

Cited by 29 publications

References 36 publications

Transposon tools hopping in vertebrates

Transposon tools hopping in vertebrates

Postintegrative Gene Silencing within the Sleeping Beauty Transposition System

Active integration: new strategies for transgenesis

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