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1 December 2001 Developmental Potential of Porcine Nuclear Transfer Embryos Derived from Transgenic Fetal Fibroblasts Infected with the Gene for the Green Fluorescent Protein: Comparison of Different Fusion/Activation Conditions
Kwang-Wook Park, Liangxue Lai, Hee-Tae Cheong, Gi-Sun Im, Qing-Yuan Sun, Guangming Wu, Bill N. Day, Randall S. Prather
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Abstract

The in vitro developmental potential of porcine nuclear transfer (NT) embryos was evaluated. Oocytes were matured for 42–44 h, and metaphase II-oocytes were enucleated. Fetal fibroblasts infected with the enhanced green fluorescent protein (EGFP) gene were serum-starved for 3–5 days. A single cell was injected into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were allocated to different fusion and activation conditions. In experiment 1, two different fusion/activation conditions were compared: two pulses of 1.2 kV/cm for 30 μsec (group A), or one pulse of 1.6 kV/cm for 30 μsec followed in 30 min by one pulse of 1.2 kV/cm for 30 μsec (group B). Parthenogenetic controls were created by using the group A parameter. The fusion rate in group A (mean ± SEM, 68.4% ± 3.9%) was higher (P < 0.05) than in group B (59.4% ± 2.3%). The rates of cleavage (50.1% ± 4.6% to 62.8% ± 5.5%) were not different among control and treatment groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% ± 3.1%) was higher (P < 0.05) than the rate of NT embryos (5.9% ± 1.7% and 4.9% ± 2.5%). In experiment 2, we compared two pulses of 1.2 kV/cm (group C) versus two pulses of 1.3 kV/cm (group D). For two control groups, the same pulses as those given to group C or D, respectively, were supplied. The fusion rate in group D (70.6% ± 4.2%) was higher (P < 0.05) than in group C (58.9% ± 2.7%). The cleavage rates were not different among control and treatment groups (58.1% ± 8.1% to 73.6% ± 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% ± 1.7%) was lower (P < 0.05) than in controls and group C (11.4% ± 2.0% to 16.4% ± 1.1%). In experiment 3, we examined whether the presence of cytochalasin B (CB) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group with CB (78.4% ± 1.4%) was higher (P < 0.05) than in the group without CB (70.9% ± 0.2%). The cleavage rate of the control group (85.5% ± 4.9%) was higher (P < 0.05) than those of the treatment groups (61.6% ± 2.7% and 63.9% ± 4.3%). However, the rates of embryos developing to the blastocyst stage (8.1% ± 2.5% to 19.1% ± 6.0%) and the mean cell number of blastocysts (29.4 ± 5.2 to 45.7 ± 6.4) were not different among control and treatment groups. Green fluorescence was observed at all stages in NT embryos. These results indicate that two pulses of 1.2 kV/cm are enough for fusion/activation of NT embryos to develop to the blastocyst stage, and that the presence of CB during donor cell injection is not necessary for early development of NT embryos.

Kwang-Wook Park, Liangxue Lai, Hee-Tae Cheong, Gi-Sun Im, Qing-Yuan Sun, Guangming Wu, Bill N. Day, and Randall S. Prather "Developmental Potential of Porcine Nuclear Transfer Embryos Derived from Transgenic Fetal Fibroblasts Infected with the Gene for the Green Fluorescent Protein: Comparison of Different Fusion/Activation Conditions," Biology of Reproduction 65(6), 1681-1685, (1 December 2001). https://doi.org/10.1095/biolreprod65.6.1681
Published: 1 December 2001
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