Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer.
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.
Koo
Ok Jae
OJ
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea.
Hossein
Mohammad Shamim
MS
Hong
So Gun
SG
Martinez-Conejero
Jose A
JA
Lee
Byeong Chun
BC
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D003260:Contact Inhibition; D003513:Cycloheximide / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004285:Dogs; D004423:Ear / Q000502:physiology*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology*; D053652:Nuclear Transfer Techniques; D011687:Purines / Q000494:pharmacology; D016192:Resting Phase, Cell Cycle / Q000187:drug effects / Q000502:physiology*; D000077546:Roscovitine; D044967:Serum
D000935:Antifungal Agents; D000970:Antineoplastic Agents; D016166:Free Radical Scavengers; D011687:Purines; D000077546:Roscovitine; D003513:Cycloheximide; D004121:Dimethyl Sulfoxide