88 CELL CYCLE SYNCHRONIZATION OF FIBROBLASTS DERIVED FROM TRANSGENIC CLONED CATTLE EAR SKIN: EFFECTS OF SERUM STARVATION, ROSCOVITINE, AND CONTACT INHIBITION

2007 ◽  
Vol 19 (1) ◽  
pp. 161
Author(s):  
X.-Z. Sun ◽  
S.-H. Wang ◽  
Y.-H. Zhang ◽  
Y.-P. Dai ◽  
N. Li
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Contact Inhibition ◽  
Developmental Competence ◽  
G1 Phase ◽  
Serum Starvation ◽  
Becton Dickinson ◽  
Cell Cycle Stage ◽  
Cell Cycle Synchronization

Cell cycle stage plays a critical role in somatic cell nuclear transfer (SCNT), and G0/G1 stage cells are preferred nuclear donors in attempts to produce cloned livestocks. Enhancement of survivability of cloned calves by roscovitine (the cyclin-dependent kinase-2 inhibitor) has been shown (Gibbons et al. 2002 Biol. Reprod. 66, 895–900). The purpose of this study was to evaluate the effects of serum starvation, roscovitine, and contact inhibition on cell cycle synchronization at the G0/G1 stage of transgenic cloned bovine ear skin-derived fibroblasts. The cell line was established from a cloned calf expressing green fluorescent protein (GFP). Data were analyzed by using SAS (8.0) with ANOVA (SAS Institute, Inc., Cary, NC, USA). At passage 2-6, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) in T-25 culture flasks from immediately after subculture until monolayer cells reached 90% confluence at 39�C, under 5% CO2 in humidified air. Then cells grown in different flasks were randomly distributed to groups: Serum starvation (SS, culture medium changed to DMEM + 0.5% FBS), roscovitine (R, cultured in DMEM + 10% FBS + 15 �M roscovitine), and contact inhibition (CI, DMEM + 10% FBS). From Day 1 to Day 5 after treatment, 3 flasks of cells from each group were subjected to fixation and staining every day, followed by determination of cell cycle stage with Becton Dickinson FACScan (Kues et al. 2000 Biol. Reprod. 62, 412–419). At least 3 replicates were performed for each determination. The results showed that in the cycling fibroblasts (50–60% confluence), 59.29% of the cells were at the G0/G1 phase. For the SS group, the proportion of G0/G1 cells was significantly higher for treatment lasting 3 days, 4 days, and 5 days than for treatment lasting 1 day or 2 days (91.5%c, 91.7%c, and 93.5%c vs. 90.1%b and 88.8%a, respectively; P < 0.05). No statistical differences were observed among 3-day, 4-day, and 5-day treatments. For the R group, one-day treatment was significantly lower in synchronizing cells to the G0/G1 stage than that from Day 2 to Day 5 (86.51%a vs. 91.1%a, 90.1%a, 89.4%a, and 91.3%b, respectively; P < 0.05) during which similar rates of G0/G1 phase were observed. For the CI group, an increase of G0/G1 stage cells was found after 3 days of CI (from 89.4%a for Day 1 and 90.4%ab for Day 2, to 91.4%bc for Day 3; P < 0.05), which was similar to 4 days (91.6%bc) and 5 days (92.1%c) for the CI group. We also found that the efficiency of obtaining G0/G1 phase cells was lower when roscovitine was employed to synchronize the cell cycle than when the SS and CI methods were used (89.7%b vs. 91.1%a and 91.0%a, respectively; P < 0.05), after analysis of the most effective treatment duration of each group. Our data indicate that in attempts to harvest adequate G0/G1-stage cells for re-cloning of transgenic cattle from fibroblasts established from ear skin of a newborn transgenic cloned calf, 3 days of treatment is enough regardless of the method (SS, R, or CI). Further research is needed to evaluate the developmental competence of embryos cloned from cells prepared by the abovementioned treatments. [a-c mean statistical differences; P < 0.05.]

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.

59 SYNCHRONIZATION OF CELL CYCLE STAGE OF BUFFALO (BUBALUS BUBALIS) FETAL FIBROBLAST CELLS BY DIFFERENT TREATMENTS

2011 ◽  
Vol 23 (1) ◽  
pp. 135
Author(s):  
N. L. Selokar ◽  
A. George ◽  
A. P. Saha ◽  
R. Sharma ◽  
M. Muzaffar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bubalus Bubalis ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Cloning Efficiency ◽  
Fetal Fibroblast ◽  
Cell Cycle Stage ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Major Factors

Cell cycle stage of donor cells significantly influences the cloning efficiency during SCNT. Donor cells in G1/G0 stage have better capability to undergo nuclear reprogramming following transfer to an unfertilized oocyte. The lack of availability of cells synchronized at G1/G0 stage is one of the major factors limiting cloning efficiency in buffalo. The aim of this study was to compare the efficacy of various methods for cell cycle synchronization of buffalo fetal fibroblast cells for SCNT. Cells isolated from fetus, 2 to 3 months old, were cultured in DMEM + 10% FBS. The primary culture was sub-cultured 8 to 10 times. For cell cycle synchronization, the cells were cultured to 1) 60 to 70% confluence (controls), 2) 60 to 70% confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (serum starved), 3), full confluence followed by culture for additional 3 to 5 days (full confluent), 4) full confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (full confluent+serum starved) and 5) 60 to 70% confluence followed by treatment with roscovitine (10, 20, or 30 μM) for 24 h. The synchronization efficiency was examined by propidium iodide staining followed by analysis of DNA content using flow cytometry and the data were analysed by 1-way ANOVA followed by Fisher’s l.s.d. test after arcsine transformation. The percentage of cells in G0/G1 phase of cell cycle was significantly higher (P < 0.05) in the full confluent+serum starved and roscovitine treated (20 or 30 μM) groups than that in the full confluent group and that treated with 10 μM roscovitine which, in turn, was higher (P < 0.05) than that in the serum starved and control groups. These results suggest that buffalo fetal fibroblast cells can be synchronized by roscovitine treatment or by serum starvation of fully confluent cell cultures to obtain a high proportion of cells in G0/G1 stage for SCNT. Table 1.Buffalo skin fibroblast cells at various stages following different treatments for cell cycle synchronization Supported by grant No. 1(5)/2007-NAIP from ICAR, India.

73 IMPROVEMENT OF CANINE CLONING EFFICIENCY BY OPTIMIZED DONOR CELL PREPARATION

2010 ◽  
Vol 22 (1) ◽  
pp. 195
Author(s):  
S. W. Park ◽  
Y. W. Jeong ◽  
J. J. Kim ◽  
K. H. Ko ◽  
S. H. Jeong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Electrical Stimulation ◽  
Somatic Cell ◽  
Somatic Cells ◽  
Contact Inhibition ◽  
Abnormal Development ◽  
Serum Starvation ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Cloned Dog

The Tibetan Mastiff is the oldest dog breed in the world, and it is at the edge of extinction. Li et al. (2008) believe that protection of and research on the Tibetan Mastiff is extremely urgent, yet few studies have been carried out, particularly at the molecular level. Somatic cell nuclear transfer (SCNT) is an efficient technique for the conservation of endangered animals because it can increase the number of individuals within a population. Considering the virtually unlimited value of cloned canids in critical biotechnology applications, including gene conservation of endangered canids and disease models, the effect of cell-cycle synchronization methods, including the use of cycling canine adult skin fibroblasts (CASF), on the cell-cycle stage and viability of donor nuclei was analyzed. To improve the efficiency of cloned dog production, optimal conditions of donor cells were analyzed by culture duration (Days 1, 2, 3, and 4), passages (2, 4, 7, 10, and 11 passages) and mitotic regulator Plk-1/-4 gene expression. Simerly et al. (2003) reported that the depletion of microtubule motors and centrosomal proteins during enucleation of SCNT procedures caused abnormal development of SCNT embryos. We therefore analyzed Plk-1/-4-induced centriole biogenesis in CASF at different passages of donor cells. In this study, somatic cells were collected from a purebred 9-month-old male Mastiff and an 11-month-old female mastiff. In vivo-matured oocytes were retrieved from outbreed dogs by operation. Cycling cells cultured at Day 4 showed a similar effect to that of cells that were artificially synchronized (contact inhibition or serum starvation). It was also confirmed that fresh and short-term culture (<5 passages) resulted in fewer harmful effects and the same cell viability as control cells, using proliferation assays and expression levels of Plk-1/-4 genes. Therefore, 4 passage-cycling cells at Day 4 were used as donor cells of SCNT. A total of 289 oocytes were reconstructed with each male or female somatic cell and then simultaneously fused/activated with 2 DC pulses of 1.9 kV cm-1 for 30 s of electrical stimulation. Finally, 224 embryos were transferred to 16 naturally synchronized recipients. As a result, we were able to use somatic cells collected from both female and male Tibetan Mastiffs to produce 10 female and 6 male mastiffs. Moreover, one surrogate delivered a quartet of identical cloned female Tibetan Mastiffs puppies; each of 3 surrogates also delivered triplets. Microsatellite analysis demonstrated the genotypic identity of the cloned puppies. In conclusion, the present study shows that (1) cell-cycle synchronization of donor cells by serum starvation/contact inhibition is not required, (2) Plk-1/-4 mRNA can be used to select the donor cells, (3) electrical stimulation alone is sufficient for the activation of SCNT embryos for the production of SCNT cloned dogs, and (4) the cloned dog delivery efficiency (7.1%) was threefold higher than in previous reports. SWP and YWJ contributed equally to this work. WSH was corresponding author and SHH was co-corresponding author.

Cloning and functional expression of a degradation-resistant novel isoform of p27Kip1

2000 ◽  
Vol 353 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Katsuya HIRANO ◽  
Mayumi HIRANO ◽  
Ying ZENG ◽  
Junji NISHIMURA ◽  
Keiichi HARA ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Growth Arrest ◽  
Negative Regulator ◽  
G1 Phase ◽  
Serum Starvation ◽  
Cell Contact ◽  
The Novel ◽  
Specific Expression

p27Kip1 is an inhibitor of cyclin-dependent kinases. It has been implicated as having a role in the induction of growth arrest at the G1 phase of the cell cycle in response to anti-mitogenic signals such as cell contact and serum starvation. Proteasome-mediated degradation plays an important role in the rapid inactivation of p27Kip1, causing quiescent cells to re-enter the cell cycle. Although the existence of a second isoform has been suggested, no such isoform was isolated. Through screening of a cDNA library derived from growth-arrested confluent porcine endothelial cells, we obtained clones for a novel isoform of p27Kip1 in addition to the original isoform. The novel isoform differed from the original isoform at the C-terminus. The tissue-specific expression of the original and novel isoforms was demonstrated at the mRNA and protein levels. An in vitro degradation assay demonstrated this novel isoform to be resistant to proteasome-mediated destruction. The expression as a fusion protein with green fluorescent protein revealed this isoform to be targeted to the nucleus by a bipartite nuclear-localization signal with a C-terminal part different from that of the original isoform. The expression of the novel isoform caused the growth arrest of HeLa cells and an accumulation of cells in the G0/G1 phase, and this effect was similar to that seen with the original isoform. The present study suggests that the novel isoform functions as a negative regulator of the cell cycle, and may play a distinct role. The novel isoform was named p27Kip1R because of its resistance to degradation.

Phospholipase Cδ1 regulates cell proliferation and cell-cycle progression from G1- to S-phase by control of cyclin E–CDK2 activity

2008 ◽  
Vol 415 (3) ◽  
pp. 439-448 ◽  
Author(s):  
Katherine A. Kaproth-Joslin ◽  
Xiangquan Li ◽  
Sarah E. Reks ◽  
Grant G. Kelley
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Cyclin E ◽  
Critical Role ◽  
S Phase ◽  
G1 Phase ◽  
Serum Starvation ◽  
Cycle Progression ◽  
Cdk2 Activity

In the present study, we examined the role of PLCδ1 (phospholipase C δ1) in the regulation of cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLCδ1, but not PLCβ3 or PLCϵ, induces a proliferation defect in Rat-1 and NIH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [3H]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G0/G1-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G1-phase but delayed entry into S-phase. Consistent with these findings, G1 cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G1-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E–CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLCδ1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLCδ1 in cell-cycle progression from G1-to-S-phase through regulation of cyclin E–CDK2 activity and p27 levels.

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.

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.

Constitutive expression of the E2F1 transcription factor in fibroblasts alters G0 and S phase transit following serum stimulation

1996 ◽  
Vol 74 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Thomas J. Logan ◽  
Kelly L. Jordan ◽  
David J. Hall
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cell Lines ◽  
Constitutive Expression ◽  
S Phase ◽  
G1 Phase ◽  
Serum Starvation ◽  
Serum Stimulation ◽  
G0 Phase ◽  
E2f1 Transcription Factor

The E2F1 transcription factor was constitutively expressed in NIH3T3 fibroblasts to determine its effect on the cell cycle. These E2F1 cell lines were not tightly synchronized in G0 phase of the cell cycle following serum starvation, as are normal fibroblasts. Instead, the cells are spread throughout G0 and G1 phase with a portion of the population initiating DNA synthesis. Upon serum stimulation, the remaining cells in G0/G1 begin to enter S phase immediately but with a reduced rate. Constitutive expression of E2F1 appears to primarily affect the G0 phase, since transit of proliferating E2F1 cell lines through G1 phase is the same as control cells. Consistent with a shortened G0 phase, the E2F1 cell lines have a significantly reduced cellular volume. Additionally, the first S phase after serum stimulation, but not subsequent S phases, is nearly doubled in the E2F1 cell lines compared with control cells. Cell lines expressing a deletion mutant of E2F1 (termed E2F1d87), known to significantly affect cell shape, have cell cycle and volume characteristics similar to the E2F1 expressing cells. However, all S phase durations are considerably lengthened and the cells demonstrate delayed growth after plating.Key words: cell cycle, E2F1 transcription factor, G0/G1 phase.

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