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

  • Ok Jae Koo (a1), Mohammad Shamim Hossein (a1), So Gun Hong (a1), Jose A. Martinez-Conejero (a2) and Byeong Chun Lee (a3)...

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.

Copyright

Corresponding author

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: bclee@snu.ac.kr

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