Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein

Lee, Gab Sang; Kim, Hye Soo; Hyun, Sang Hwan; Lee, So Hyun; Jeon, Hyun Yong; Nam, Dong Hyun; Jeong, Yeon Woo; Kim, Sue; Kim, Ji Hye; Han, Jae Yong; Ahn, Curie; Kang, Sung Keun; Lee, Byeong Chun; Hwang, Woo Suk

doi:10.1016/j.theriogenology.2004.04.017

Cited by 64 publications

(46 citation statements)

References 52 publications

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“…After being washed twice with Hepes-TLP-PVA (Tyrode-lactate-pyruvate-polyvinyl alcohol) and the maturation medium, respectively, COCs with compact cumulus cells and evenly granulated ooplasm were used for the experiments. COCs (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) were cultured in a droplet of maturation medium (200 µl) under paraffin oil (Nacalai Tesque, Kyoto, Japan) in a polystyrene dish (35-mm: Becton Dickinson Labware, Oxnard, CA, USA) and cultured at 38.5 C in an atmosphere of 5% CO 2 in air. The maturation medium was TCM-199 with Earle's salts (Gibco, BRL, Grand Island, NY, USA) supplemented with 0.91 mM sodium pyruvate (Sigma-Aldrich Chemical, St. Louis, MO, USA), 3.05 mM glucose (Wako Pure Chemical Industries, Osaka, Japan), 0.57 mM cysteine hydrochloride hydrate (Sigma), 10 ng/ml epidermal growth factor (Sigma), 10 IU/ml eCG (ASKA Pharmaceutical, Tokyo, Japan), 10 IU/ml hCG (ASKA), 100 µg/ml amikacin sulfate (Meiji Seika, Tokyo, Japan) and 10% (v/v) pig follicular fluid.…”

Section: In Vitro Maturation Of Oocytesmentioning

confidence: 99%

“…Several reports have shown that CB treatment of SCNT embryos resulted in increased blastocyst formation in vitro [18][19][20][21]. However, it has been reported that postactivation treatment with CB had no effect on either blastocyst rate or cell number of SCNT embryos [9,22]. CB possess cytotoxicity [23], and its cytotoxicity might have a detrimental effect on the subsequent development of SCNT embryos.…”

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confidence: 99%

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Effect of Postactivation Treatment with Latrunculin A on In Vitro and In Vivo Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

Himaki

Mizobe

Tsuda

et al. 2012

Journal of Reproduction and Development

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Abstract. The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term. Key words: Aged donor, Clawn miniature pig, Cytoskeletal inhibitors, Latrunculin A, Nuclear transfer (J. Reprod. Dev. 58: [398][399][400][401][402][403] 2012) C lawn miniature pigs (inbred strain) have a potential value for biomedical research and xenotransplantation due to their clear genetic background [1][2][3] and few individual differences; thus, they have stimulated investigation of novel strategies directed at generating a genetically modified miniature pig by somatic cell nuclear transfer (SCNT). We have shown how to produce live offspring derived from Clawn miniature pig SCNT embryos; however, the efficiency still remains low [4]. To date, pigs have been cloned with SCNT using donor cells derived from fetal pigs to pigs several years of age. The efficiency of SCNT is influenced by many factors, including quality of recipient oocytes, type of donor cells, reprogramming of donor nuclei and condition of recipient gilts [4][5][6][7][8][9][10][11][12][13]. In particular, the diploid complement retention is one of the most important factors in improving the developmental ability of SCNT embryos [14,15]. SCNT embryos derived from diploid cells are generally treated with cytoskeletal inhibitors, such as cytochalasin B (CB), after activation to prevent extrusion of a pseudo polar body (pPB) and the possible loss of chromosomes maintain normal ploidy [7,16,17]. Several reports have shown that CB treatment of SCNT embryos resulted in increased blastocyst formation in vitro [18][19][20][21]. However, it has been reported that postactivation treatment with CB had no effect on either blastocyst rate or cell number of SCNT embryos [9,22]. CB possess cytotoxicity [23], and its cytotoxicity might have a detrimental effect on the subsequent development of SCNT embryos.Latrunculin A (LatA), a toxin purified from the Red Sea sponge Latrunculia magnifica, is a member of the cytoskel...

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Section: In Vitro Maturation Of Oocytesmentioning

confidence: 99%

mentioning

confidence: 99%

Effect of Postactivation Treatment with Latrunculin A on In Vitro and In Vivo Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

Himaki

Mizobe

Tsuda

et al. 2012

Journal of Reproduction and Development

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“…54: [156][157][158][159][160][161][162][163] 2008) n recent years, techniques for producing cloned pigs by somatic cell nuclear transfer (SCNT) have been actively utilized to produce genetically modified pigs. A number of cloned pigs have been produced, including those with genes for GFP [1][2][3][4][5][6][7][8], as well as alpha1,3-galactosyltransferase knockout pigs [9][10][11][12][13][14][15][16]. In this manner, genetically modified pigs are being used in a wide variety of biomedical fields, ranging from basic research to organ transplantation.…”

mentioning

confidence: 99%

Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

Kurome

Ishikawa

Tomii

et al. 2008

Journal of Reproduction and Development

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Abstract. Miniature pigs have been recognized as valuable experimental animals in various fields such as medical and pharmaceutical research. However, the amount of information on somatic cell cloning in miniature pigs, as well as genetically modified miniature pigs, is much less than that available for common domestic pigs. The objective of the present study was to establish an efficient technique of cloning miniature pigs by somatic cell nuclear transfer. A high pregnancy rate was achieved following transfer of parthenogenetic (3/3) and cloned (5/6) embryos using female miniature pigs in the early pregnancy period as recipients after estrus synchronization with prostaglandin F2 alpha analog and gonadotrophins. The production efficiency of the cloned miniature pigs using male and female fetal fibroblasts as nucleus donors was 0.9% (2/215 and 3/331, respectively). Cloned miniature pigs were also produced efficiently (7.8%, 5/64) by transferring reconstructed embryos into the uteri of common domestic pigs. When donor cells transfected with the green fluorescent protein (GFP) gene were used in nuclear transfer, the production efficiency of the reconstructed embryos and rate of blastocyst development were comparable to those obtained by non-transfected cells. When transfected cell-derived reconstructed embryos were transferred to three common domestic pig recipients, all became pregnant, and a total of ten transgenic cloned miniature pigs were obtained (piglet production efficiency: 2.7%, 10/365). Hence, we were able to establish a practical system for producing cloned and transgenic-cloned miniature pigs with a syngeneic background. Key words: Embryo transfer, In vitro matured oocyte, Miniature pig, Nuclear transfer, Transgenic (J. Reprod. Dev. 54: [156][157][158][159][160][161][162][163] 2008) n recent years, techniques for producing cloned pigs by somatic cell nuclear transfer (SCNT) have been actively utilized to produce genetically modified pigs. A number of cloned pigs have been produced, including those with genes for GFP [1][2][3][4][5][6][7][8], as well as alpha1,3-galactosyltransferase knockout pigs [9][10][11][12][13][14][15][16]. In this manner, genetically modified pigs are being used in a wide variety of biomedical fields, ranging from basic research to organ transplantation.To date, more than ten breeds of miniature pigs have been established as experimental and companion animals [17]. As far as the use of pigs as experimental animals is concerned, miniature pigs are smaller and easier to handle than common domestic pigs, and they have been used in a variety of fields, such as medical and pharmacological research [10,[18][19][20][21][22][23][24][25]. However, compared with common domestic pigs, far less information regarding the production of cloned and genetically modified pigs is available for miniature pigs [10,13,14,[26][27][28][29][30][31].In the present study, we conducted a series of experiments including (i) validation of an estrus synchronization procedure for miniature pig recipients, (i...

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“…These products include different biopharmaceuticals/therapeutic proteins such as: 1) human haemoglobin in porcine erythrocytes (Swanson et al 1992, Sharma et al 1994 and 2) human blood coagulation/clotting factor VIII in milk (Paleyanda et al 1997). They also involve: 3) human blood clotting factor IX and additional recombinant isoforms of porcine lactoferrin simultaneously secreted to milk as well as 4) human erythropoietin in urine (Lee et al 2005).…”

Section: Perspectives Of Somatic Cell Cloning and Transgenesis Of Pigmentioning

confidence: 99%

“…Nonetheless, Lee et al (2005) have generated male and female transgenic cloned piglets, in which the expression of fusion protein consisting of recombinant human erythropoietin (rhEPO) and eGFP-reporter protein was driven under the promoter (target-ing vector) of porcine UPII gene into the urinary tract (bladder). This indicates that it is also possible to produce genetically-transformed individuals excreting biopharmaceuticals in the urine not only in laboratory animals such as mice, but also in large animals such as farm livestock species (Fig.…”

Section: Introductionmentioning

confidence: 99%

The possibilities of practical application of transgenic mammalian species generated by somatic cell cloning in pharmacology, veterinary medicine and xenotransplantology

Samiec

Skrzyszowska

2011

Polish Journal of Veterinary Sciences

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Cloning of genetically-modified mammals to produce: 1) novel animal bioreactors expressing human genes in rens, urinary bladder and the male accessory sex glands, as well as 2) porcine organs suitable in pig-to-human xenotransplantology, could offer new advantages for biomedical purposes. So too does the generation and/or multiplication of genetically-engineered cloned animals in order to produce: 3) physiologically-relevant animal models of serious monogenic human diseases and 4) prion disease-resistant small as well as large animals (i.e., rodents, ruminants). The basic purpose of this paper is to overview current knowledge deciphering the possibilities of using transgenic specimens created by somatic cell nuclear transfer in medical pharmacology, veterinary medicine, agriculture, transplantational medicine and immunology.

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Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein

Cited by 64 publications

References 52 publications

Effect of Postactivation Treatment with Latrunculin A on <i>In Vitro</i> and <i>In Vivo</i> Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

Effect of Postactivation Treatment with Latrunculin A on <i>In Vitro</i> and <i>In Vivo</i> Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

The possibilities of practical application of transgenic mammalian species generated by somatic cell cloning in pharmacology, veterinary medicine and xenotransplantology

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