title: Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer.
abstract: 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.
authors.affiliation: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea.
authors.forename: Ok Jae
authors.initials: OJ
authors.lastname: Koo
authors.forename: Mohammad Shamim
authors.initials: MS
authors.lastname: Hossein
authors.forename: So Gun
authors.initials: SG
authors.lastname: Hong
authors.forename: Jose A
authors.initials: JA
authors.lastname: Martinez-Conejero
authors.forename: Byeong Chun
authors.initials: BC
authors.lastname: Lee
mesh_terms: 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
chemical_list: D000935:Antifungal Agents; D000970:Antineoplastic Agents; D016166:Free Radical Scavengers; D011687:Purines; D000077546:Roscovitine; D003513:Cycloheximide; D004121:Dimethyl Sulfoxide