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

The in vitro developmental potential of porcine nuclear transfer (NT) embry os was evaluated. Oocytes were matured for 42-44 h, and metaphase II-oocyte s were enucleated. Fetal fibroblasts infected with the enhanced green fluor escent protein (EGFP) gene were serum-starved for 3-5 days. A single cell w as injected into the perivitelline space of the enucleated oocytes. The rec onstructed oocytes were allocated to different fusion and activation condit ions. In experiment 1, two different fusion/activation conditions were comp ared: two pulses of 1.2 kV/cm for 30 mu sec (group A), or one pulse of 1.6 kV/cm for 30 mu sec followed in 30 min by one pulse of 1.2 kV/cm for 30 mu sec (group B). Parthenogenetic controls were created by using the group A p arameter. The fusion rate in group A (mean +/- SEM, 68.4% +/- 3.9%) was hig her (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 treatme nt groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% +/- 3.1%) was higher (P < 0.05) than the rat e of NT embryos (5.9% +/- 1.7% and 4.9% +/- 2.5%). In experiment 2, we comp ared two pulses of 1.2 kV/cm (group Q 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 st age in group D (3.5% +/- 1.7%) was lower (P < 0.05) than in controls and gr oup C (11.4% +/- 2.0% to 16.4% +/- 1.1%). In experiment 3, we examined whet her the presence of cytochalasin B (CO) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group wit h CB (78.4% +/- 1.4%) was higher (P < 0.05) than in the group without CB (7 0.9% +/- 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 6 3.9% +/- 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 blastoc ysts (29.4 +/- 5.2 to 45.7 +/- 6.4) were not different among control and tr eatment 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 devel opment of NT embryos.