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

Ma L, Liu X, Wang F, He X, Chen S, Li W - PLoS ONE (2015)

Bottom Line: 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.However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability.

View Article: PubMed Central - PubMed

Affiliation: School of Mathematics, Physics and Biological Engineering, Inner Mongolia University of Science & Technology, Baotou, Inner Mongolia, China.

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.

No MeSH data available.


Related in: MedlinePlus

In Vitro Cultured Cloned Sheep Embryos.A, 2- and 8-cell embryos; B, early blastocyst; C, hatching blastocyst; D, early blastocyst stained with Hoechst 33342. Scale bar: 20 μm.
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pone.0135344.g004: In Vitro Cultured Cloned Sheep Embryos.A, 2- and 8-cell embryos; B, early blastocyst; C, hatching blastocyst; D, early blastocyst stained with Hoechst 33342. Scale bar: 20 μm.

Mentions: After culture using different methods, fibroblasts were used as nuclear donors for SCNT. The cloned embryos could develop to blastocysts in 7–8 days and the blastocysts hatched in 9–10 days (Fig 4). The developmental ability of the cloned embryos in the present study was comparable with the results of our and other previous studies [18, 23, 24], and the results are shown in Table 3.


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

Ma L, Liu X, Wang F, He X, Chen S, Li W - PLoS ONE (2015)

In Vitro Cultured Cloned Sheep Embryos.A, 2- and 8-cell embryos; B, early blastocyst; C, hatching blastocyst; D, early blastocyst stained with Hoechst 33342. Scale bar: 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4546374&req=5

pone.0135344.g004: In Vitro Cultured Cloned Sheep Embryos.A, 2- and 8-cell embryos; B, early blastocyst; C, hatching blastocyst; D, early blastocyst stained with Hoechst 33342. Scale bar: 20 μm.
Mentions: After culture using different methods, fibroblasts were used as nuclear donors for SCNT. The cloned embryos could develop to blastocysts in 7–8 days and the blastocysts hatched in 9–10 days (Fig 4). The developmental ability of the cloned embryos in the present study was comparable with the results of our and other previous studies [18, 23, 24], and the results are shown in Table 3.

Bottom Line: 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.However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability.

View Article: PubMed Central - PubMed

Affiliation: School of Mathematics, Physics and Biological Engineering, Inner Mongolia University of Science & Technology, Baotou, Inner Mongolia, China.

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.

No MeSH data available.


Related in: MedlinePlus