Abstract
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.
Highlights
In the technology of somatic cell nuclear transfer (SCNT), a differentiated somatic nucleus is transferred into the cytoplasm of a mature, enucleated oocyte; the cytoplasm of the oocyte has the ability to reprogram the somatic nucleus into a totipotent state
Somatic cells arrested in G0/G1 phase were recommended as the ideal nuclear donors in SCNT because they facilitated coordination in the cell cycle of the somatic nuclei and the cytoplasm of oocytes [6, 7]
During SCNT, if the nuclear genetic material was totally removed from an oocyte, and a somatic cell in G0/G1 phase was injected into this enucleated oocyte, after this reconstructed embryo was activated, and a certain protein synthesis inhibitor was used to prevent the exclusion of genetic material, this SCNT-derived embryo would have the correct number of chromosomes [8]
Summary
In the technology of somatic cell nuclear transfer (SCNT), a differentiated somatic nucleus is transferred into the cytoplasm of a mature, enucleated oocyte; the cytoplasm of the oocyte has the ability to reprogram the somatic nucleus into a totipotent state. The efficiency of SCNT technology is low, and it can be influenced by many factors, such as the quality of the mature oocytes [1], the type, passage number and cell cycle phase of the donor cells [2,3,4], and the procedure used for SCNT [5]. Two methods, serum starvation or culture to full confluency, have been used to arrest somatic cells in G0/G1 phase, so that they can be used as nuclear donors in SCNT [7, 9,10,11]. If somatic cells are subjected to one of these two methods and used as nuclear donors in SCNT, it is unclear whether the resulting cloned embryos are comparable in developmental ability and quality
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