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Production of Nuclear Transfer-Derived Piglets Using Porcine Fetal Fibroblasts Transfected with the Enhanced Green Fluorescent Protein1"

Cited 123 time in Web of Science Cited 136 time in Scopus
Authors
Hyun, Sanghwan; Lee, Gabsang; Kim, Daeyoung; Kim, Hyesoo; Lee, Sohyun; Nam, Donghyun; Jeong, Yeonwoo; Kim, Sue; Yeom, Soocheong; Kang, Sungkeun; Han, Jae Yong; Lee, Byeongchun; Hwang, Woosuk
Issue Date
2003
Publisher
Society for the Study of Reproduction
Citation
Biology of Reproduction, vol.69 no.3, pp. 1060-1068
Keywords
early developmentembryogamete biologyovary
Abstract
A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1) received a simultaneous electrical fusion/ activation (S-EFA or G-EFA groups), or 2) were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3) were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P , 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%–69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P , 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P , 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE- 6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%–69%) were higher than those with gilt oocytes (23%–27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFFSCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.
ISSN
0006-3363
Language
English
URI
https://hdl.handle.net/10371/100197
DOI
https://doi.org/10.1095/biolreprod.102.014886
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Food and Animal Biotechnology (식품·동물생명공학부)Journal Papers (저널논문_식품·동물생명공학부)
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