Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

Abstract

The present study was conducted to examine the effect of cell culture conditions, antioxidants, protease inhibitors (PI), and different levels of dimethylsulfoxide (DMSO) for the promotion of synchronization of different cell cycles of Siberian tiger skin fibroblasts. We also compared the ability of somatic cell nuclei of the Siberian tiger in pig cytoplasts and to support early development after reconstruction. Cell cycle synchronization between nuclear donor and recipient cells is considered to be one of the most crucial factors for successful cloning. Five experiments were performed each with a one‐way completely randomized design involving three replicates of all treatments. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused in the effects of cycling, serum starved and fully confluent stages of Siberian tiger cells on different cell cycles. In Experiment II, the effects of different antioxidants like β‐Mercaptoethanol (β‐ME, 10 µM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 4 hr. In Experiment III, three PI, namely 6‐dimethylaminopurine (6‐DMAP, 2 mM), cycloheximide (7.5 µg/ml) and cytochalasin B (7.5 µg/ml) were used in the sane manner as in Experiment II. In Experiment IV, different levels of DMSO at 0%, 0.5%, 1.0%, and 2.5% were tested on different cell cycle stages of Siberian tiger examined by Flowcytometry (FACS). In Experiment I, 67.2% of the Siberian tiger skin fibroblasts reached the G₀/G₁ stage (2C DNA content) in fully confluent conditions which was more than the cycling (49.8%) and serum starved (SS) medium (65.5%; P < 0.05). Among the chemically treated group, glutathione (72.6%) and cycloheximide (71.3%) had little bit better results for the synchronization of G₀ + G₁ phases than serum starved and fully confluent. After nuclear transfer we did not see any significant differences on the development of tiger‐porcine reconstructed embryos at cycling, SS and fully confluent. Data indicate that prolonged culture of cells in the absence of serum as well as using different chemicals for this experiment does not imply a shift in the percentage of cells that enter G₀/G₁ and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs in Siberian tiger, because there are negative effects, such as apoptosis associated with serum starvation. Mol. Reprod. Dev. 74: 403–411, 2007.