A Novel Method for the Production of Transgenic Cloned Pigs: Electroporation-Mediated Gene Transfer to Non-Cultured Cells and Subsequent Selection with Puromycin1

Watanabe, Satoshi; Iwamoto, Masao; Suzuki, Shunichi; Fuchimoto, Daiichiro; Honma, Daisuke; Nagai, Takashi; Hashimoto, Michiko; Yazaki, Satoko; Sato, Masahiro; Ōnishi, Akira

doi:10.1095/biolreprod.104.031591

Cited by 52 publications

(41 citation statements)

References 23 publications

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“…Transgenic pigs expressing fluorescent cell markers have been generated by many research groups [7,8,27,[65][66][67][68][69][70][71][72][73][74][75][76]. For example, Webster et al [72] produced transgenic pigs that simultaneously expressed red fluorescent protein from Discosoma (DsRed), enhanced GFP (EGFP) and enhanced blue fluorescent protein (EBFP).…”

Section: Development Of Transgenic Pigs With Reporter Genesmentioning

confidence: 99%

Application of Genetically Modified and Cloned Pigs in Translational Research

Matsunari

Nagashima

2009

Journal of Reproduction and Development

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Abstract. Pigs are increasingly being recognized as good large-animal models for translational research, linking basic science to clinical applications in order to establish novel therapeutics. This article reviews the current status and future prospects of genetically modified and cloned pigs in translational studies. It also highlights pigs specially designed as disease models, for xenotransplantation or to carry cell marker genes. Finally, use of porcine somatic stem and progenitor cells in preclinical studies of cell transplantation therapy is also discussed. Key words: Cloned pig, Gene knockout, Transgenic pig, Translational research (J. Reprod. Dev. 55: [225][226][227][228][229][230] 2009) t present, pigs are not only considered important livestock but are also essential large-animal models in various types of biomedical research [1][2][3][4][5][6]. Furthermore, due to recent technological advances in genetic modification and somatic cell cloning, the range of applications for porcine models has increased markedly [7][8][9][10][11][12][13][14]. The present review focuses on development of genetically modified and cloned pigs and their use in translational studies.Translational research plays an important role as a bridge between basic science outcomes and the clinical realm, leading to development of novel therapeutics. However, disparate findings for rodent models and humans have hindered the translation of rodent data into actionable technologies for patients [15]. In contrast, pigs have many anatomical and physiological similarities to humans, including their cardiovascular systems, omnivorous gastrointestinal tracts and central nervous systems. Their physical sizes are also more comparable to that of humans, rendering pigs more appropriate for development of new surgical or endoscopic techniques.In the future, more advanced translational research may be conducted by improving the clinical relevance of pig models through genetic engineering. This article reviews the current status and future prospects of genetically modified pigs in preclinical studies of stem and progenitor cell therapy, the development of disease models and xenotransplantation.

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Section: Development Of Transgenic Pigs With Reporter Genesmentioning

confidence: 99%

Application of Genetically Modified and Cloned Pigs in Translational Research

Matsunari

Nagashima

2009

Journal of Reproduction and Development

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“…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%

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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“…Previous studies have suggested that one explanation for the failure to detect an antibiotic-resistant gene after selection is the so called 'by-stander effect' [4,7]; that is, expression of an antibiotic resistance gene in some cells could confer antibiotic resistance to nearby cells either by direct contact between cells or by secretion of the gene product into the medium. However, Watanabe et al [6] have reported that puromycin can be used for selection of recombinant cells from non-cultured cells.…”

Section: Discussionmentioning

confidence: 99%

“…The difference between the two groups' observations was the donor cells used; Betthauser et al [7] used crossbred (Yorkshire/Landrace/ Berkshire cross)-derived donor cells, whereas Watanabe et al [6] used Landrace-derived donor cells. We used adult earderived fibroblast cells from a gnotobiotic miniature pig and four commercial pigs, including three inbred (Duroc, Landrace and Berkshire) pigs and one crossbred pig (Duroc × Landrace × Berkshire).…”

Section: Discussionmentioning

confidence: 99%

“…Although the use of somatic cell donors for nuclear transfer in livestock offers many advantages over pronuclear microinjection, previous studies [4,5] have shown that selection of transgenic cells using G418 was not sufficient to completely remove the nontransgenic cells from cultures, and scNT blastocysts as well as cloned animals derived from the donor cells were not always transgenic. As an alternative method to G418 selection, Watanabe et al [6] showed that puromycin can be effectively used to select transgenic cells. However, it has also been reported that after puromycin selection, some nontransgenic cells are still puromycin-resistant, as reported for Betthauser et al [7].…”

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confidence: 99%

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Production of Transgenic Pigs Harboring the Human Erythropoietin (hEPO) Gene Using Somatic Cell Nuclear Transfer

Cho¹,

Hwang

Choi

et al. 2009

Journal of Reproduction and Development

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Abstract. The production of transgenic pigs using somatic cell nuclear transfer (scNT) has been widely described, but a technique for removing nontransfected donor cells and for creating different founder animals has not yet been fully elucidated. In this study, four different expression vectors (pBC1hEPO, pMARBC1hEPO, pBC1hEPOwpre and pMARBC1hEPOwpre) were compared to determine the highest transgene expression, ideal conditions of enrichment of recombinant cells in vitro and efficiency of transgenesis following transfection into HC11 mammary epithelial cells. The highest protein expression in HC11 cells was obtained from the pMARBC1hEPOwpre expression vector. Next, we evaluated the efficiency of transgenic pig production by using geneticin (G418) selection alone or by using real-time PCR selection following G418 selection. Ideal enrichment of recombinant cells was obtained by a combination of realtime PCR and G418 selection; embryos reconstructed using donor cells selected by a combination of real-time PCR and G418 selection gave rise to nine piglets, all of which were transgenic. Among them, three founder transgenic pigs were established. Exogenous DNA fragments were shown to be integrated into chromosomes 1q2.4, 1p2.3 and 6q2.4, respectively, in these three pigs. However, the transgenic rate using G418 selection alone was only 33% (two of six pigs) and showed a very low efficacy compared with that of the combination of real-time PCR and G418 selection. Our results provide a valuable experimental model for applying and evaluating transgenic technology in pigs. Key words: Erythropoietin, Geneticin (G418), Pig, Somatic cell nuclear transfer (SCNT), Transgenic (J. Reprod. Dev. 55: [128][129][130][131][132][133][134][135][136] 2009) enetic manipulation, including transfer of foreign DNA into somatic cells and selection of transgenic somatic cells, is a crucial aspect of producing transgenic animals by somatic cell nuclear transfer (scNT). The low efficiency of scNT livestock production is a major obstacle to widespread use of this technology for production of transgenic animals. Most commercial companies producing transgenic livestock use scNT extensively only to produce transgenic founders. Microinjection of DNA into the pronuclei of fertilized oocytes has been the only practical means of producing transgenic livestock since the method was established in 1985 [1]. However, only a small proportion (approximately 5%) of animals integrate the exogenous DNA into their genome. Many transgenic lines do not provide sufficiently high levels of transgene expression because the integration site is random. Recently, the scNT technique has provided an alternative method for pronuclear microinjection as a means of transferring exogenous DNA to the germ line of an animal that allows for precise genetic modifications by gene targeting [2,3]; Schnieke et al. [2] demonstrated that production of transgenic animals using the scNT technique improved overall efficiency to 2.5 times that of pronuclear microinjection. These previou...

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A Novel Method for the Production of Transgenic Cloned Pigs: Electroporation-Mediated Gene Transfer to Non-Cultured Cells and Subsequent Selection with Puromycin1

Cited by 52 publications

References 23 publications

Application of Genetically Modified and Cloned Pigs in Translational Research

Application of Genetically Modified and Cloned Pigs in Translational Research

Pig transgenesis by Sleeping Beauty DNA transposition

Production of Transgenic Pigs Harboring the Human Erythropoietin (hEPO) Gene Using Somatic Cell Nuclear Transfer

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