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Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer

Published online by Cambridge University Press:  01 February 2009

Ok Jae Koo ,
Mohammad Shamim Hossein ,
So Gun Hong ,
Jose A. Martinez-Conejero  and
Byeong Chun Lee
Ok Jae Koo
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea.
Mohammad Shamim Hossein
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea.
So Gun Hong
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea.
Jose A. Martinez-Conejero
Affiliation:
Fundacion IVI, Instituto Universitario IVI, Valencia University, Valencia, Spain.
Byeong Chun Lee*
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151–742, Korea.
*
All correspondence to: Byeong Chun Lee. Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151–742, Korea. Tel: +822 880 1269. Fax: +822 873 1269. e-mail: [email protected]
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Summary

Cycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents – dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) – on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

Keywords

CanineCell cycle synchronizationEar fibroblastRoscovitineSomatic cell nuclear transfer
Type
Research Article
Information
Zygote , Volume 17 , Issue 1 , February 2009 , pp. 37 - 43
Copyright
Copyright © Cambridge University Press 2008

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