title: Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein.
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 PFF-SCNT 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.
authors.affiliation: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea.
authors.forename: Sanghwan
authors.initials: S
authors.lastname: Hyun
authors.forename: Gabsang
authors.initials: G
authors.lastname: Lee
authors.forename: Daeyoung
authors.initials: D
authors.lastname: Kim
authors.forename: Hyesoo
authors.initials: H
authors.lastname: Kim
authors.forename: Sohyun
authors.initials: S
authors.lastname: Lee
authors.forename: Donghyun
authors.initials: D
authors.lastname: Nam
authors.forename: Yeonwoo
authors.initials: Y
authors.lastname: Jeong
authors.forename: Sue
authors.initials: S
authors.lastname: Kim
authors.forename: Soocheong
authors.initials: S
authors.lastname: Yeom
authors.forename: Sungkeun
authors.initials: S
authors.lastname: Kang
authors.forename: Jaeyong
authors.initials: J
authors.lastname: Han
authors.forename: Byeongchun
authors.initials: B
authors.lastname: Lee
authors.forename: Woosuk
authors.initials: W
authors.lastname: Hwang
mesh_terms: D000367:Age Factors; D000704:Analysis of Variance; D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D019976:Cloning, Organism / Q000379:methods*; D004624:Embryo Transfer / Q000662:veterinary*; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D015571:Follicular Fluid / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000378:metabolism*; D006080:Ovarian Follicle / Q000166:cytology / Q000378:metabolism; D011247:Pregnancy; D013552:Swine / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins