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In vitro developmental competence of pig nuclear transferred embryos: effects of GFP transfection, refrigeration, cell cycle synchronization and shapes of donor cells

Published online by Cambridge University Press:  01 August 2006

Yun-Hai Zhang ,
Deng-Ke Pan ,
Xiu-Zhu Sun ,
Guo-Jie Sun ,
Xiao-Hui Liu ,
Xiao-Bo Wang ,
Xing-Hua Tian ,
Yan Li ,
Yun-Ping Dai  and
Ning Li
Yun-Hai Zhang
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Deng-Ke Pan
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China. College of Animal Science and Technology, China Agricultural University, Beijing 100094, P. R. China.
Xiu-Zhu Sun
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Guo-Jie Sun
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Xiao-Hui Liu
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Xiao-Bo Wang
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Xing-Hua Tian
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China. College of Animal Science and Technology, China Agricultural University, Beijing 100094, P. R. China.
Yan Li
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Yun-Ping Dai
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Ning Li*
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
*
All correspondence to: Ning Li, State Key Laboratory for Agrobiotechnology, China Agricultural University, No. 2 Yuan-ming-yuan west Road, Haidian, Beijing 100094, P. R. China. Tel: +86 10 62731146. Fax: +86 10 62733904. e-mail: [email protected]
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Summary

The present study was designed to evaluate the feasibility of producing pig transgenic blastocysts expressing enhanced green fluorescent protein (GFP) and to examine the effects of shape and preparation methods of donor cells on in vitro developmental ability of pig nuclear transferred embryos (NTEs). In experiment 1, the effect of GFP transfection on development of pig NTEs was evaluated. The cleavage and blastocyst rates showed no significant difference between NTEs derived from transfected and non-transfected donors. In experiment 2, the effect of different nuclear donor preparation methods on in vitro development of NTEs was examined. The cleavage rate showed no statistically significant differences among three preparation methods. The blastocyst rates of donor cells treated once at −4 °C and those of freshly digested cells were similar to each other (26.3% vs 17.9%). The lowest blastocyst rates (5.88%) were observed when cells cryopreserved at −196 °C were used as donors. In experiment 3, the effect of different cell cycle synchronization methods on the in vitro development potential of pig NTEs was evaluated. The cleavage rate of NTEs derived from cycling cells was much better than that of NTEs derived from serum-starved cells (64.4% vs 50.5%, p < 0.05), but no significant difference was observed between the the blastocyst rates of the two groups. In experiment 4, the effect of different shapes of cultured fibroblast cells on the in vitro development of pig NTEs was examined. The fusion rate for couplets derived from rough cells was poorer than that observed in couplets derived from round smooth cells (47.8% vs 76.8%, p < 0.05). However, there were no significant differences observed in the cleavage rate and blastocyst rate. In conclusion, the present study indicated that (i) refrigerated pig GFP-transfected cells could be used as donors in nuclear transfer and these NTEs could be effectively developed to blastocyst stage; (ii) serum starvation of GFP-transfected cells is not required for preimplantation development of pig NTEs; and (iii) a rough surface of GFP-transfected donor cells affects fusion rate negatively but has no influence on the cleavage rate or blastocyst rate of pig NTEs.

Keywords

Cell refrigerationGFPPigPreimplantational developmentSomatic nuclear transfer
Type
Research Article
Information
Zygote , Volume 14 , Issue 3 , August 2006 , pp. 239 - 247
Copyright
Copyright © Cambridge University Press 2006

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