Transgenic Pigs Produced Using in Vitro Matured Oocytes Infected With a Retroviral Vector

Cabot, Ryan; Kühholzer, B.; Chan, Anthony W.S.; Lai, Liangxue; Park, K.-W.; Chong, Kowit-Yu; Schatten, Gerald; Murphy, Clifton N.; Abeydeera, Lalantha R.; Day, Bill; Prather, Randall S.

doi:10.1081/abio-100108347

Cited by 96 publications

(62 citation statements)

References 12 publications

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“…The overall results confirm the statements on the absence of differences between wildtype pigs and pigs expressing GFP in previous studies (Li et al 2009;Brunetti et al 2008;Kurome et al 2006;Webster et al 2005;Cabot et al 2001) and experience at the Roslin Institute. The comparatively high number of animals and the broadness of parameters observed, however, put conclusions on the absence of detrimental effects of the transgene expression in question on the welfare of the animals involved on a more robust and verifiable basis.…”

Section: Discussionsupporting

confidence: 90%

“…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. However, these assumptions were based on anecdotal observations in relatively few animals.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

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

et al. 2011

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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%

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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“…Retroviral vectors based on Moloney murine leukaemia virus transfer genes efficiently into murine, porcine and bovine embryos (Cabot et al, 2001;Chan et al, 1998;Jaenisch, 1976); however, retroviruses are subject to epigenetic modification, and retroviral expression is shut off during embryogenesis ( Jaenisch, 1976) or shortly after birth (Chan et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Efficient transgenesis in farm animals by lentiviral vectors

Hofmann¹,

Keßler²,

Sonja³

et al. 2003

EMBO Rep

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1054Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germline. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.

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Transgenic Pigs Produced Using in Vitro Matured Oocytes Infected With a Retroviral Vector

Cited by 96 publications

References 12 publications

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

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

Pig transgenesis by Sleeping Beauty DNA transposition

Efficient transgenesis in farm animals by lentiviral vectors

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