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

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

Research output: Contribution to journalArticle

Abstract

This study was performed to develop a system for porcine somatic cell nuclear transfer (SCNT) and to produce human erythropoietin (hEPO)-transgenic cloned piglets. Porcine fetal fibroblasts were transfected with an expression plasmid (phEPO-GFP). In Experiment 1, the effect of transfection of phEPO-GFP transgene on development of porcine SCNT embryos was investigated. Three fetal fibroblast cell lines (two male and one female) with or without transfected with phEPO-GFP trasngene were used as donor cells for SCNT. Lower fusion rates were observed in two lines of transfected cells as compared to those of the control cells. In Experiment 2, the effect was examined of elevated Ca2+ concentration in the fusion/activation medium on development of transfected SCNT embryos. The rates of fusion and blastocyst formation were significantly increased by supplementing 1.0 mM of CaCl2 (versus 0.1 mM) into the fusion/activation medium. In Experiment 3, the effect was studied of a chemical treatment (cytochalasin B) after electric fusion/activation (F/A) on porcine transgenic SCNT embryo development. The electric F/A + cytochalasin B treatment increased total cell number in blastocysts as compared to that of electric F/A treatment alone. In Experiment 4, transgenic cloned embryos were transferred to surrogate mothers and a total of six cloned piglets were born. Transgenic cloned piglets were confirmed by polymerase chain reaction and Southern blot analysis. From a single surrogate mother, female and male transgenic cloned piglets were produced by transferring pooled SCNT embryos derived from female and male transfected donor cells. In conclusion, a system for porcine SCNT was developed and led to the successful production of hEPO transgenic cloned piglets.

Original languageEnglish (US)
Pages (from-to)973-991
Number of pages19
JournalTheriogenology
Volume63
Issue number4
DOIs
StatePublished - Mar 1 2005
Externally publishedYes

Fingerprint

Green Fluorescent Proteins
green fluorescent protein
somatic cells
fibroblasts
piglets
Fibroblasts
genetically modified organisms
embryo transfer
swine
erythropoietin
Swine
cytochalasin B
Embryo Transfer
blastocyst
Surrogate Mothers
Cytochalasin B
cell lines
cells
Blastocyst
Erythropoietin

Keywords

  • Embryos
  • EPO
  • Nuclear transfer
  • Porcine
  • Reproductive technologies
  • Topic category form
  • Transgenic

ASJC Scopus subject areas

  • Small Animals
  • Food Animals
  • Animal Science and Zoology
  • Equine

Cite this

Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein. / Lee, Gabsang; 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.

In: Theriogenology, Vol. 63, No. 4, 01.03.2005, p. 973-991.

Research output: Contribution to journalArticle

Lee, G, Kim, HS, Hyun, SH, Lee, SH, Jeon, HY, Nam, DH, Jeong, YW, Kim, S, Kim, JH, Han, JY, Ahn, C, Kang, SK, Lee, BC & Hwang, WS 2005, 'Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein', Theriogenology, vol. 63, no. 4, pp. 973-991. https://doi.org/10.1016/j.theriogenology.2004.04.017
Lee, Gabsang ; 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. / Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein. In: Theriogenology. 2005 ; Vol. 63, No. 4. pp. 973-991.
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abstract = "This study was performed to develop a system for porcine somatic cell nuclear transfer (SCNT) and to produce human erythropoietin (hEPO)-transgenic cloned piglets. Porcine fetal fibroblasts were transfected with an expression plasmid (phEPO-GFP). In Experiment 1, the effect of transfection of phEPO-GFP transgene on development of porcine SCNT embryos was investigated. Three fetal fibroblast cell lines (two male and one female) with or without transfected with phEPO-GFP trasngene were used as donor cells for SCNT. Lower fusion rates were observed in two lines of transfected cells as compared to those of the control cells. In Experiment 2, the effect was examined of elevated Ca2+ concentration in the fusion/activation medium on development of transfected SCNT embryos. The rates of fusion and blastocyst formation were significantly increased by supplementing 1.0 mM of CaCl2 (versus 0.1 mM) into the fusion/activation medium. In Experiment 3, the effect was studied of a chemical treatment (cytochalasin B) after electric fusion/activation (F/A) on porcine transgenic SCNT embryo development. The electric F/A + cytochalasin B treatment increased total cell number in blastocysts as compared to that of electric F/A treatment alone. In Experiment 4, transgenic cloned embryos were transferred to surrogate mothers and a total of six cloned piglets were born. Transgenic cloned piglets were confirmed by polymerase chain reaction and Southern blot analysis. From a single surrogate mother, female and male transgenic cloned piglets were produced by transferring pooled SCNT embryos derived from female and male transfected donor cells. In conclusion, a system for porcine SCNT was developed and led to the successful production of hEPO transgenic cloned piglets.",
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