title: Effect of fusion/activation protocol on in vitro development of porcine nuclear transfer embryos constructed with foreign gene-transfected fetal fibroblasts.
abstract: The effect of fusion/activation protocol on in vitro development of porcine nuclear transfer (NT) embryos constructed with foreign gene-transfected somatic cells were investigated. NT embryos were produced by using enucleated M II oocytes and enhanced green fluorescence protein (EGFP) gene-transfected or non-transfected porcine fetal fibroblasts. One group of NT embryos received a single electrical pulse to induce fusion and activation simultaneously (FAS). The other group was fused 2 hr before activation (FBA) using two kinds of electrical pulses. Electrically activated NT embryos in both groups were treated with cycloheximide (CHX) before culture to assess the development to the blastocyst stage. After 6 days of culture, all morulae and blastocysts derived from EGFP-transfected fibroblasts emitted green fluorescence without mosaicism, and EGFP-gene product was also detected in all morulae and blastocysts examined. NT embryos undergoing FAS showed higher developmental capacity to blastocysts than those undergoing FBA, regardless of the EGFP transfection into the nuclear donor cells. The results also indicated that EGFP-gene transfection into nuclear donor cells has no obvious deleterious effect on the development of NT embryos to blastocysts.
authors.affiliation: Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
authors.forename: Mario A
authors.initials: MA
authors.lastname: Martinez Diaz
authors.forename: Tadashi
authors.initials: T
authors.lastname: Mori
authors.forename: Masashi
authors.initials: M
authors.lastname: Nagano
authors.forename: Seiji
authors.initials: S
authors.lastname: Katagiri
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002459:Cell Fusion; D004624:Embryo Transfer / Q000662:veterinary*; D005260:Female; D005347:Fibroblasts / Q000502:physiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology*; D013552:Swine; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins