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Cell-cycle synchronization of fibroblasts derived from transgenic cloned cattle ear skin: effects of serum starvation, roscovitine and contact inhibition

Published online by Cambridge University Press:  01 May 2008

XiuZhu Sun ,
ShuHui Wang ,
YunHai Zhang ,
HaiPing Wang ,
LiLi Wang ,
Liu Ying ,
Rong Li  and
Ning Li
XiuZhu Sun
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China.
ShuHui Wang
Affiliation:
Institute of Animal Science Chinese Academy of Agricultural Science Beijing100094, China.
YunHai Zhang
Affiliation:
AnHui Agricultural University, HeFei, 230036, AnHui province, China.
HaiPing Wang
Affiliation:
Beijing GenProtein Biotechnology Ltd, BeiJing, 100094, China.
LiLi Wang
Affiliation:
Beijing GenProtein Biotechnology Ltd, BeiJing, 100094, China.
Liu Ying
Affiliation:
Beijing GenProtein Biotechnology Ltd, BeiJing, 100094, China.
Rong Li
Affiliation:
Beijing GenProtein Biotechnology Ltd, BeiJing, 100094, China.
Ning Li*
Affiliation:
State Key Laboratory for Agrobiotechnology, College of Biological Science China Agricultural University, Beijing 100094, China. State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China.
*
All correspondence to: Ning Li. State Key Laboratory for Agrobiotechnology, College of Biological Science China Agricultural University, Beijing 100094, China. Tel: +86 10 62733323. Fax: +86 10 62733904. e-mail: [email protected]
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Summary

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.

Keywords

Cell-cycle synchronizationFlow cytometry Somatic cell nuclear transferTransgenic
Type
Research Article
Information
Zygote , Volume 16 , Issue 2 , May 2008 , pp. 111 - 116
Copyright
Copyright © Cambridge University Press 2008

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