Cell-cycle synchronization of fibroblasts derived from transgenic cloned cattle ear skin: effects of serum starvation, roscovitine and contact inhibition.
The purpose of the present study was to evaluate the effects of serum-starvation, contact-inhibition and roscovitine treatments on cell-cycle synchronization at the G0/G1 stage of ear skin fibroblasts isolated from transgenic cloned cattle. The developmental competence of re-cloned embryos was also examined. Our results showed that the proportion of G0/G1 cells from the serum-starved group at 3, 4 or 5 days was significantly higher compared with 1 or 2 days only (91.5, 91.7 and 93.5% versus 90.1 and 88.8%, respectively, p < 0.05); whilst there was no statistical difference among cells at 3, 4 or 5 days. For roscovitine-treated cells, the proportion of G0/G1 cells at 2, 3, 4 or 5 days was significantly higher than those treated for 1 day only (91.1, 90.1, 89.4 and 91.3% versus 86.51%, respectively, p < 0.05). The proportion of contact-inhibited G0/G1 cells rose significantly with treatment time, but was similar at 3, 4 and 5 days (89.4, 90.4, 91.4, 91.6 and 92.1%, respectively, p < 0.05). The efficiency of obtaining G0/G1 phase cells was lower when roscovitine treatment was employed to synchronize the cell cycle compared with the serum-starvation and contact-inhibition methods (89.7 versus 91.1% and 91.0%, p < 0.05). Moreover, obvious differences were observed in the rate of fused couplets and blastocysts (89.88 +/- 2.70 versus 87.40 +/- 5.13; 44.10 +/- 8.62 versus 58.38 +/- 13.28, respectively, p < 0.05), when nuclear transfer embryos were reconstructed using donors cells that had been serum starved or contact inhibited for 3 days. Our data indicate that 3 day treatment is feasible for harvesting sufficient G0/G1 cells to produce re-cloned transgenic bovine embryos, regardless of whether serum-starvation, contact-inhibition or roscovitine treatments are used as the synchronization methods.
Sun
Xiuzhu
X
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China.
Wang
Shuhui
S
Zhang
Yunhai
Y
Wang
Haiping
H
Wang
Lili
L
Ying
Liu
L
Li
Rong
R
Li
Ning
N
D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle / Q000235:genetics*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D019976:Cloning, Organism; D003260:Contact Inhibition; D005347:Fibroblasts / Q000166:cytology*; D053652:Nuclear Transfer Techniques; D009865:Oocytes; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D044967:Serum
D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine