Production of Nuclear Transfer-Derived Swine That Express the Enhanced Green Fluorescent Protein

Park, Kwang-Wook; Cheong, Hyeonsook; Lai, Liangxue; Im, Gi‐Sun; Kühholzer, B.; Bonk, Aaron J.; Samuel, Melissa; Rieke, August; Day, Billy N.; Murphy, Clifton N.; Carter, D. B.; Prather, Randall S.

doi:10.1081/abio-100108344

Cited by 174 publications

(139 citation statements)

References 16 publications

(13 reference statements)

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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%

“…Permanent genetic modification of somatic donor cells for SCNT has been achieved by random genomic insertion of plasmid DNA (Hyun et al 2003;Kragh et al 2009;Kragh et al 2004;Watanabe et al 2005) or by genomic integration of transduced retroviral or lentiviral vectors Park et al 2001;Park et al 2002). In both non-viral and viral approaches inclusion of a drug resistance gene in the transgene-encoding vectors allows selection for cells containing the integrated vector.…”

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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“…The cloning of sheep (7) and subsequently pigs (8)(9)(10) by nuclear transfer with somatic cells has made attempts to knockout the GGTA1 locus in pigs technically feasible.…”

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confidence: 99%

Production of α-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations

Kolber-Simonds

Lai

Watt

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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417

338

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Hyperacute rejection of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-␣-1,3-galactose (Gal␣-1,3-Gal) epitopes expressed on the pig cell surface. Previously, we generated inbred miniature swine with a null allele of the ␣-1,3-galactosyltransferase locus (GGTA1) by nuclear transfer (NT) with gene-targeted fibroblasts. To expedite the generation of GGTA1 null pigs, we selected spontaneous null mutant cells from fibroblast cultures of heterozygous animals for use in another round of NT. An unexpectedly high rate of spontaneous loss of GGTA1 function was observed, with the vast majority of null cells resulting from loss of the WT allele. Healthy piglets, hemizygous and homozygous for the genetargeted allele, were produced by NT by using fibroblasts that had undergone deletional and crossover͞gene conversion events, respectively. Aside from loss of Gal␣-1,3-Gal epitopes, there were no obvious phenotypic differences between these null piglets and WT piglets from the same inbred lines. In fact, congenital abnormalities observed in the heterozygous NT animals did not reappear in the serially produced null animals.A ntibodies against galactosyl-␣-1,3-galactose (Gal␣-1,3-Gal) residues on cell surface glycoproteins of pig cells mediate hyperacute rejection of porcine organs in primate model recipients and are the most immediate barrier to successful clinical xenotransplantation (1, 2). High levels of preformed ''natural'' antibodies against the Gal␣-1,3-Gal epitope are found in humans and old world primates, following evolutionary loss of the corresponding galactosyltransferase activity (encoded by GGTA1) (3). The presence of these antibodies, along with the high density of Gal␣-1,3-Gal residues on most pig cells (4), suggests that elimination of GGTA1 function would provide a practical means of overcoming both hyperacute rejection and subsequent acute or chronic tissue damage associated with antibody binding to this epitope.The lack of GGTA1 function in humans and old world primates, along with the viability of GGTA1 knockout mice produced with embryonic stem cell technology (5, 6), suggested that a knockout strategy might be biologically feasible in pigs. The cloning of sheep (7) and subsequently pigs (8-10) by nuclear transfer with somatic cells has made attempts to knockout the GGTA1 locus in pigs technically feasible.We have previously reported the generation of GGTA1 heterozygous inbred miniature swine using nuclear transfer with gene-targeted fibroblasts (11). Starting with heterozygous fibroblasts from such animals, we now report the isolation of GGTA1 null cells with spontaneous loss of the WT allele. The rate of loss of heterozygosity (LOH) was several orders of magnitude greater than typically expected, an observation that may be related to the inbred background of the heterozygous animals. LOH resulted in some cases from deletion of the WT allele and in others from either somatic crossing over or gene conversion. Similarly high rates of somatic recombi...

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“…Nuclear Transfer (NT) was carried out using the procedure described by Park et al (2001) with a slight modification. For nuclear transfer, the cumulus-free oocytes matured in vitro were enucleated by aspirating the first polar body and the adjacent cytoplasm using fine glass pipette.…”

Section: Nuclear Transfermentioning

confidence: 99%

Coordinated change of a ratio of methylated H3-Iysine 4 or acetylated H3 to acetylated H4 and DNA methylation is associated with tissue-specific gene expression in cloned pig

Kang

Park²,

Chung³

et al. 2007

Exp Mol Med

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Various cell types in higher multicellular organisms are genetically homogenous, but are functionally and morphologically heterogeneous due to the differential expression of genes during development, which appears to be controlled by epigenetic mechanisms. However, the exact molecular mechanisms that govern the tissue-specific gene expression are poorly understood. Here, we show that dynamic changes in histone modifications and DNA methylation in the upstream coding region of a gene containing the transcription initiation site determine the tissue-specific gene expression pattern. The tissue-specific expression of the transgene correlated with DNA demethylation at specific CpG sites as well as significant changes in histone modifications from a low ratio of methylated H3-lysine 4 or acetylated H3-lysine 9, 14 to acetylated H4 to higher ratios. Based on the programmed status of transgene silenced in cloned mammalian ear-derived fibroblasts, the transgene could be reprogrammed by change of histone modification and DNA methylation by inhibiting both histone deacetylase and DNA methylation, resulting in high expression of the transgene. These findings indicate that dynamic change of histone modification and DNA methylation is potentially important in the establishment and maintenance of tissue-specific gene expression.

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Production of Nuclear Transfer-Derived Swine That Express the Enhanced Green Fluorescent Protein

Cited by 174 publications

References 16 publications

Pig transgenesis by Sleeping Beauty DNA transposition

Pig transgenesis by Sleeping Beauty DNA transposition

Production of α-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations

Coordinated change of a ratio of methylated H3-Iysine 4 or acetylated H3 to acetylated H4 and DNA methylation is associated with tissue-specific gene expression in cloned pig

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