Efficient transgenesis in farm animals by lentiviral vectors

Hofmann, Andreas; Keßler, Barbara; Sonja, Ewerling; Weppert, Myriam; Vogg, Barbara; Ludwig, Harald; Stojković, Miodrag; Boelhauve, Marc; Brem, Gottfried; Wolf, Eckhard; Pfeifer, Alexander

doi:10.1038/sj.embor.7400007

Cited by 245 publications

(209 citation statements)

References 20 publications

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“…Retro-and lentiviral vectors are established potent tools for stable modification of porcine somatic donor cells for SCNT Park et al 2001;Park et al 2002) and have been successfully used for transgene delivery to porcine embryos (Cabot et al 2001;Hofmann et al 2003;Whitelaw et al 2004). SB-directed transgenesis combined with SCNT and HMC represents a nonviral alternative that offers insertion of a defined genetic unit, strong systemic transgene expression, and possibilities of multicopy transgene insertion.…”

Section: Discussionmentioning

confidence: 99%

“…Transgenesis in pigs has previously been achieved by transferring the transgene to oocytes or early embryos by pronuclear injection of foreign DNA (Hammer et al 1985;Hirabayashi et al 2001;Nottle et al 2001), transduction with retro-and lentiviral vectors injected into the perivitelline space of the oocyte (Cabot et al 2001;Hofmann et al 2003;Whitelaw et al 2004) or, alternatively, by sperm-mediated gene transfer (Naruse et al 2005;Webster et al 2005). With the successful cloning of animals by somatic cell nuclear transfer (SCNT), it is now possible to produce transgenic pigs from genetically engineered somatic donor cells.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Pig transgenesis by Sleeping Beauty DNA transposition

Jakobsen

Kragh

et al. 2010

Transgenic Res

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“…In addition to PNI and SCNT, lentiviral transduction of zygotes 24,25 and intracytoplasmatic sperm injection (ICSI) 26,27 have been employed for transgenesis in the pig. Lentiviral transduction can produce high ratios of transgenic offspring (20-30 % transgenic offspring per treated embryos), but this technique frequently results in cell mosaicism and thus reduced germline transmission; the maximal cargo of foreign DNA with lentiviral vectors is about 7 kbp; and epigenetic silencing of the virally integrated constructs has been observed 13 .…”

Section: Germline Transgenesis In Pigsmentioning

confidence: 99%

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

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2The pig has emerged as an important large animal model in biomedical and pharmaceutical research.We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer, and offers comparable efficacies to lentiviral approaches, without limitations on vector design, issues of transgene silencing as well as the toxicity and biosafety concerns of working with viral vectors. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in one day; generation of germline-transgenic lines by using this protocol takes approximately one year.

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“…Hofmann et al 2003;Li et al 2009;Whitelaw et al 2004) where it allows to visualize gene expression real time and in vivo. It has been considered to be ´non-invasive´ (Hadjantonakis and Nagy 2001) and transgenic pigs expressing GFP have been described as normal (Li et al 2009;Brunetti et al 2008;Kurome et al 2006;Webster et al 2005) and healthy (Cabot et al 2001) before.…”

Section: Introductionmentioning

confidence: 99%

Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP)

et al. 2011

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Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology -animal welfare -has not been approached through systematic assessment and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals along various stages of post natal development. The protocol used covered reproductory performance and behaviour in GFP and wildtype sows and general health and development, social behaviour, exploratory behaviour and emotionality in GFP and wildtype littermates from birth until an age of roughly four months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs expressing GFP as healthy. Although the results are not surprising in the light of previous experience, they give a more solid fundament to the evaluation of GFP expression as being relatively non-invasive in pigs. The present study may furthermore serve as starting point for researchers aiming at a systematic characterization of welfare relevant effects in the line of transgenic pigs they are working with. AbstractSince large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology -animal welfare -has not been approached through systematic assessment and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs.The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals along various stages of post natal development. The protocol used covered reproductory performance and behaviour in GFP and wildtype sows and general health and development, social behaviour, exploratory behaviour and emotionality in GFP and wildtype littermates from birth until an age of roughly four months.The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs expressing GFP as healthy. Although the results are not surprising in the light of previous experience, they give a more solid fundament to the evaluation of GFP expression as being relatively non-invasive in pigs...

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Efficient transgenesis in farm animals by lentiviral vectors

Cited by 245 publications

References 20 publications

Pig transgenesis by Sleeping Beauty DNA transposition

Pig transgenesis by Sleeping Beauty DNA transposition

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP)

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