Full confluency, serum starvation, and roscovitine for inducing arrest in the G₀/G₁ phase of the cell cycle in puma skin-derived fibroblast lines

Abstract

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G₀/G₁ of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G₀/G₁ (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P > 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G₀/G₁ (P > 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G₀/G₁ stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.

Keywords:
felids; cell cycle synchronization; culture to confluence; somatic cell nuclear transfer