piggyBac
 is a flexible and highly active transposon as compared to
 Sleeping Beauty
 ,
 Tol2
 , and
 Mos1
 in mammalian cells

Wu, Sareina Chiung‐Yuan; Meir, Yaa Jyuhn James; Coates, Craig J.; Handler, Alfred M.; Pelczar, Pawel; Moisyadi, Stefan; Kaminski, Joseph

doi:10.1073/pnas.0606979103

Cited by 321 publications

(367 citation statements)

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“…One potential limitation of transposases is that instead of reaching a plateau in transposition with increasing transposase, transposon integration declines due to overproduction inhibition. We have observed overproduction inhibition with PB and it might also occur with Tol2 if the ratio of helper to donor plasmid was increased (Wu et al 2006). In contrast, Wilson and colleagues did not demonstrate overproduction inhibition with PB (Wilson et al 2007).…”

Section: Active Transgenesismentioning

confidence: 53%

“…These results suggest that the Gal4-UAS limits the number of target sites at which integration can occur, likely due to the tethering of the transposase close to the UAS target (Maragathavally et al 2006). Mos1 does not appear to be functional in mammalian systems (Wu et al 2006). However, the PB transposase coupled to the GAL4 DNA binding domain retains transposition activity similar to the wild-type, unlike Tol2 or SB.…”

Section: Mechanisms To Improve Specificity and Efficiency Of Transfecmentioning

confidence: 95%

“…To determine which DNA transposase encoding plasmids may have the greatest affect on tg insertion, four commonly used transposon transfection systems were studied in four different mammalian cell lines (Wu et al 2006), three of which were human. These initial experiments performed with the two plasmid system (Donor and Helper plasmids) demonstrated that piggyBac (PB), a transposase isolated from the cabbage looper moth Trichoplusia ni, and found to exhibit activity in a variety of species ranging from planarian to mammalian cells (Lobo et al 2006), is the most effective mediator for stable insertion of tg's in all cell lines tested.…”

Section: Active Transgenesismentioning

confidence: 99%

“…As PB is the most efficient transposon in mammalian systems (Wu et al 2006), studies to modify PB to increase its specificity and transposition efficiency are in progress. Until recently the PB literature described the transposition machinery as a two-component system: a Helper plasmid Fig.…”

Section: Mechanisms To Improve Specificity and Efficiency Of Transfecmentioning

confidence: 99%

“…We have tried to alter uC31 recombinase by coupling it to a DNA binding domain to target a specific pseudo-site but this resulted in loss of activity. In contrast, we have altered the PB transposase to direct integration and it has retained full activity (Wu et al 2006).…”

Section: Mechanisms To Improve Specificity and Efficiency Of Transfecmentioning

confidence: 99%

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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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Section: Active Transgenesismentioning

confidence: 53%

Section: Mechanisms To Improve Specificity and Efficiency Of Transfecmentioning

confidence: 95%

Section: Active Transgenesismentioning

confidence: 99%

Section: Mechanisms To Improve Specificity and Efficiency Of Transfecmentioning

confidence: 99%

Section: Mechanisms To Improve Specificity and Efficiency Of Transfecmentioning

confidence: 99%

See 3 more Smart Citations

Active integration: new strategies for transgenesis

Shinohara

Kaminski²,

Segal

et al. 2007

Transgenic Res

Self Cite

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Manipulating and studying gene function in human pluripotent stem cell models

Balmas,

Sozza,

Bottini

et al. 2023

FEBS Letters

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Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss‐, gain‐, and change‐of‐function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single‐cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.

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