scholarly journals Production of Cloned Pigs by Nuclear Transfer of Preadipocytes Following Cell Cycle Synchronization by Differentiation Induction

2009 ◽  
Vol 55 (2) ◽  
pp. 121-127 ◽  
Author(s):  
Ryo TOMII ◽  
Mayuko KUROME ◽  
Naohiro WAKO ◽  
Takashi OCHIAI ◽  
Hitomi MATSUNARI ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Differentiation Induction ◽  
Cell Cycle Synchronization

scholarly journals 69 PRODUCTION OF CLONED PIGS BY NUCLEAR TRANSFER OF PREADIPOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 184
Author(s):  
R. Tomii ◽  
M. Kurome ◽  
H. Ueda ◽  
S. Ueno ◽  
K. Hiruma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Annexin V ◽  
The Other ◽  
Serum Starvation ◽  
Male Adult ◽  
Differentiation Induction ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Fetal Fibroblasts

Since the first success in producing cloned pigs, donor cells have been limited to fetal fibroblasts and a few other cell types. The aim of the present study was to determine if porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (NT) in pigs. Preadipocytes established from subcutaneous adipose tissue of a male adult pig were used as nuclear donor cells. Cell cycle synchronization was carried out by serum starvation (5 days), confluency (5 days), roscovitine treatment (15 μM, 2 days), or differentiation induction by 0.5 mM 3-Isobutyl-1-methylxanthine, 0.25 μM dexamethasone, and 5 μg/mL insulin (5 days). Cell cycle synchronization and apoptosis of the donor cells were examined by flow cytometry and Annexin V staining and TUNEL. IVM oocytes were obtained from abattoir ovaries and matured in NCSU23. Donor cells were fused with the enucleated recipient oocytes by a single DC pulse of 200 V/mm for 10 μs in 0.28 M mannitol + 0.15 mM MgSO4. Reconstructed embryos were electrically activated at 1–1.5 h after the NT, followed by cytochalasin B treatment for 3 h. Development of the NT embryos was assessed by fixation/staining at 3 h after NT, culture for 7 days in NCSU23, and transfer to the oviducts of estrus-synchronized recipient gilts. The cells immediately entered the G0 phase by differentiation induction (92.5 ± 0.4%), with higher efficiency of synchronization than for the other methods (roscovitine: 80.3 ± 0.2%; confluency: 79.9 ± 0.3%, P < 0.05) except for serum starvation (89.8 ± 0.6%). The proportion of apoptotic cells in the differentiation group was significantly lower than the other groups (Annexin V: 7.7% vs. 15.7 to 19.3%, TUNEL: 8.3% vs. 12.8 to 14.0%, P < 0.05). Incidence of premature chromosome condensation following NT (88.0%) was as high as that observed after NT with fetal fibroblasts previously (data not shown). In vitro developmental rates of the NT embryos did not differ significantly among the cell cycle synchronization methods of the donor cells (7.2 to 10.8%). Cell number of the blastocysts was highest in the differentiation group (49.0 vs. 30.2 to 41.9, P < 0.05). Transfer of 1004 cloned embryos of the serum starvation group to 5 recipients resulted in the production of 4 live and 1 stillborn piglets from 1 recipient. Transfer of cloned embryos reconstructed of donor cells treated by differentiation induction is currently underway. These data demonstrate that preadipocytes collected from an adult pig are promising nuclear donor cells for pig cloning. This study was supported by PROBRAIN.

Cell-cycle synchronization of fibroblasts derived from transgenic cloned cattle ear skin: effects of serum starvation, roscovitine and contact inhibition

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 111-116 ◽  
Author(s):  
XiuZhu Sun ◽  
ShuHui Wang ◽  
YunHai Zhang ◽  
HaiPing Wang ◽  
LiLi Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Statistical Difference ◽  
Treatment Time ◽  
Day Treatment ◽  
Contact Inhibition ◽  
Developmental Competence ◽  
Serum Starvation ◽  
Bovine Embryos ◽  
Cell Cycle Synchronization

SummaryThe purpose of the present study was to evaluate the effects of serum-starvation, contact-inhibition and roscovitine treatments on cell-cycle synchronization at the G0/G1 stage of ear skin fibroblasts isolated from transgenic cloned cattle. The developmental competence of re-cloned embryos was also examined. Our results showed that the proportion of G0/G1 cells from the serum-starved group at 3, 4 or 5 days was significantly higher compared with 1 or 2 days only (91.5, 91.7 and 93.5% versus 90.1 and 88.8%, respectively, p < 0.05); whilst there was no statistical difference among cells at 3, 4 or 5 days. For roscovitine-treated cells, the proportion of G0/G1 cells at 2, 3, 4 or 5 days was significantly higher than those treated for 1 day only (91.1, 90.1, 89.4 and 91.3% versus 86.51%, respectively, p < 0.05). The proportion of contact-inhibited G0/G1 cells rose significantly with treatment time, but was similar at 3, 4 and 5 days (89.4, 90.4, 91.4, 91.6 and 92.1%, respectively, p < 0.05). The efficiency of obtaining G0/G1 phase cells was lower when roscovitine treatment was employed to synchronize the cell cycle compared with the serum-starvation and contact-inhibition methods (89.7 versus 91.1% and 91.0%, p < 0.05). Moreover, obvious differences were observed in the rate of fused couplets and blastocysts (89.88 ± 2.70 versus 87.40 ± 5.13; 44.10 ± 8.62 versus 58.38 ± 13.28, respectively, p < 0.05), when nuclear transfer embryos were reconstructed using donors cells that had been serum starved or contact inhibited for 3 days. Our data indicate that 3 day treatment is feasible for harvesting sufficient G0/G1 cells to produce re-cloned transgenic bovine embryos, regardless of whether serum-starvation, contact-inhibition or roscovitine treatments are used as the synchronization methods.

Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Ok Jae Koo ◽  
Mohammad Shamim Hossein ◽  
So Gun Hong ◽  
Jose A. Martinez-Conejero ◽  
Byeong Chun Lee
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Contact Inhibition ◽  
Population Doubling ◽  
Serum Starvation ◽  
Population Doubling Time ◽  
Cell Cycle Synchronization ◽  
Significant Difference

SummaryCycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents – dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) – on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

Effect of cell cycle synchronization on porcine nuclear transfer

1999 ◽  
Vol 51 (1) ◽  
pp. 215 ◽  
Author(s):  
P.J Verma ◽  
Z.T Du ◽  
C.G Grupen ◽  
R.J Ashman ◽  
S.M Mcllfatrick ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Cell Cycle Synchronization

Effects of donor sex and different cell treatments on development of yak–bovine interspecies nuclear transfer embryos

2008 ◽  
Vol 5 (1) ◽  
pp. 55-60
Author(s):  
Liu Ying ◽  
Zhu Shi-En ◽  
Li Rong ◽  
Wang Li-Li ◽  
Wang Hai-Ping ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Cell Group ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Significant Difference ◽  
Interspecies Nuclear Transfer

AbstractThe purpose of this study was to evaluate the effects of donor sex, treatments of cell cycle synchronization and donor nuclei obtained from fresh or frozen–thawed conditions on developmental competence of yak–bovine interspecies nuclear transfer embryos. Bovine (Bos taurus) oocytes were used as recipients and yak (Bos grunniens) ear fibroblast cells were used as donors. Results indicated that the development rate of male blastocysts was higher than that of female (56.6% versus 39.5%, P<0.05), whereas cleavage and total cell number showed no difference between the two groups. No significant difference was observed in the development and quality of blastocysts with donor cells treated by serum starvation or contact inhibition, and there was no significant difference in embryo development with fresh or frozen–thawed donor cells, whereas the cleavage rate in the group of frozen–thawed cells was significantly lower than that of the fresh cell group (54.5% versus 78.2%, P<0.05). The results demonstrated that donor sex could impact the developmental competence of yak–bovine interspecies nuclear transfer embryos, whereas different treatments of cell cycle synchronization and freezing had little influence.

Cell Cycle Synchronization of the Murine EO771 Cell Line Using Double Thymidine Block Treatment

BioEssays ◽  
2020 ◽  
Vol 42 (9) ◽  
pp. 1900116
Author(s):  
Marie Goepp ◽  
Delphine Le Guennec ◽  
Adrien Rossary ◽  
Marie‐Paule Vasson
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Synchronization
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