Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos

• Published in: PloS one 2015-08, Vol.10 (8), p.e0135344-e0135344
• Main Authors: Ma, LiBing, Liu, XiYu, Wang, FengMei, He, XiaoYing, Chen, Shan, Li, WenDa
• Format: Article
• Language: English
• Subjects: Animals; Bioengineering; Cell culture; Cell Culture Techniques - methods; Cell Culture Techniques - veterinary; Cell cycle; Cell Cycle Checkpoints - genetics; Cloning; Cloning, Organism - methods; Cytoplasm; Embryonic Development - genetics; Embryonic Development - physiology; Embryos; Engineering; Fibroblasts; Fibroblasts - cytology; Flow cytometry; G1 phase; G1 Phase - genetics; Gene expression; Genes; Growth factors; Kinases; Mathematics; Nuclear transfer; Nuclear Transfer Techniques; Ovis aries; Physics; Resting Phase, Cell Cycle - genetics; Reverse transcription; Science; Sheep; Sheep - embryology; Sheep - genetics; Sheep - physiology; Skin; Somatic cell nuclear transfer; Somatic cells; Synergistic effect; Viability
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0135344

It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT). Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1) cultured to 70-80% confluency (control group), (2) cultured to full confluency, (3) starved in low serum medium for 4 d, or (4) cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in G0/G1 phase, and the short-term treatment of nuclear donor cells with these two methods could improve the efficiency of SCNT.