Efficient Transposition of the piggyBac (PB) Transposon in Mammalian Cells and Mice

Ding, Sheng; Wu, Xiaohui; Li, Gang; Han, Min; Zhuang, Yuan; Xu, Tian

doi:10.1016/j.cell.2005.07.013

Cited by 866 publications

(893 citation statements)

References 39 publications

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“…17,19,44,45 The PiggyBac transposon has been reported to be capable of moving DNA elements as large as 100kb in mammalian genomes, 46 and large elements up to 14kb are transposed without apparent loss of efficiency, 47 whereas SleepingBeauty transposition efficiency drastically decreases with size. 48 These observations are consistent with Figure 7.…”

Section: Discussionmentioning

confidence: 99%

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Waldmeier

Hellmann

Gutknecht

et al. 2016

mAbs

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In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.

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Section: Discussionmentioning

confidence: 99%

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Waldmeier

Hellmann

Gutknecht

et al. 2016

mAbs

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“…PB and Tol2 have been found to be able to carry a larger cargo as compared to Sleeping Beauty (SB). For example, the PB helper can be large (9.3-14.3 Kb) without significant reduction in transposition efficiency (Ding et al 2005). Transposon systems have many attractive features as vectors for gene delivery, such as: (a) accommodating large tg; (b) being non-viral, they do not induce an immune response in rodent models; (c) inexpensive to mass produce (Kaminski and Summers 2003) and (d) mediating efficient tg integration which is stable and shows persistent expression (Ivics et al 1997).…”

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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“…SB has also been used for germ line mutagenesis but has been less effective in this context (Dupuy et al, 2001;Fischer et al, 2001;Horie et al, 2001). Recently a new transposon, PiggyBac (PB) isolated from the cabbage looper moth Trichoplusia ni, has been shown to be highly efficient for germ line insertional mutagenesis in mice (Ding et al, 2005). In contrast to SB, PB did not appear to exhibit local hopping in this context (Ding et al, 2005); however, a systematic comparison of transposition rates and genome coverage has not been reported.…”

mentioning

confidence: 99%

Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons

Liang

Kong

Stalker

et al. 2009

Genesis

133

123

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Summary: The Sleeping Beauty and PiggyBac DNA transposon systems have recently been developed as tools for insertional mutagenesis. We have compared the chromosomal mobilization efficiency and insertion site preference of the two transposons mobilized from the same donor site in mouse embryonic stem (ES) cells under conditions in which there were no selective constraints on the transposons' insertion sites. Compared with Sleeping Beauty, PiggyBac exhibits higher transposition efficiencies, no evidence for local hopping and a significant bias toward reintegration in intragenic regions, which demonstrate its utility for insertional mutagenesis. Although Sleeping Beauty had no detectable genomic bias with respect to insertions in genes or intergenic regions, both Sleeping Beauty and PiggyBac transposons displayed preferential integration into actively transcribed loci. genesis 47: [404][405][406][407][408] 2009.

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Efficient Transposition of the piggyBac (PB) Transposon in Mammalian Cells and Mice

Cited by 866 publications

References 39 publications

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Active integration: new strategies for transgenesis

Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons

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