scholarly journals 40 CHARACTERIZATION OF EARLY G1 CELLS AS NUCLEAR DONORS FOR SOMATIC CELL CLONING IN CATTLE

2005 ◽  
Vol 17 (2) ◽  
pp. 170
Author(s):  
A. Kasamatsu ◽  
K. Saeki ◽  
T. Tamari ◽  
K. Shirouzu ◽  
S. Taniguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Somatic Cell ◽  
Cell Cloning ◽  
Proliferating Cells ◽  
Blastocyst Development ◽  
Ki 67 ◽  
Quiescent Cells ◽  
Reconstructed Embryos ◽  
G1 Cells

In somatic cell cloning, the cell cycle phase of the donor cells has critical impact on nuclear reprogramming and chromosomal normality of the reconstructed embryos. Recently, enhanced development to full term was obtained with embryos reconstructed with bovine fibroblasts soon after cell division (early G1 cells, Kasinathan P et al. 2001 Nat. Biotech. 19, 1176–1178; Urakawa M et al. 2004 Theriogenology 62, 714–728). In this study, to investigate the detailed cell cycle characteristics and gene expression of the early G1 cells as nuclear donors, we examined the cell proliferating and nuclear activity by detecting PCNA and Ki-67 in the cells, and the gene expression in the cells transfected with the luciferase gene. Bovine fibroblasts were transfected with chicken β-actin/firefly luciferase fusion gene (β-act/luc+), and stably transfected; cloned cells were used for cell analysis. We compared cell cycle characteristics for quiescent cells (0.4% serum for 7 days), cell doublets (early G1 cells) prepared by the “shake-off” method, and proliferating (30 to 40% confluency) cells. The presence of PCNA and Ki-67 and the incorporation of BrdU in the cells were determined by immunohistochemical analysis. The LUC+ signal (luminescence) in the cells was detected with an imaging photon counter for 10 consecutive min. Embryos reconstructed with these cells were cultured for 168 h for examination of blastocyst development. Experiments were repeated three times, and the data were analyzed with Fisher's PLSD test following ANOVA. Incorporation of BrdU was observed only in proliferating cells (24% of the cells). Neither PCNA nor Ki-67 signals were detected in the quiescent cells. PCNA was detected but Ki-67 was not detected in early G1 cells. Both PCNA and Ki-67 were detected in the proliferating cells. A strong LUC+ signal (6354 ± 673 pixels/cell) was detected in the proliferating cells, and weak signals were detected in the early G1 (2044 ± 303 pixels/cell, P < 0.05) and quiescent cells (617 ± 59 pixels/cell, P < 0.05). The rate of blastocyst development with early G1 cells was higher (45/133, 32%) than that with starved and proliferating cells (47/233, 21%, and 41/258, 14%, respectively, P < 0.05). These results indicate that early G1 cells were actively proliferating cells because of the positive PCNA signals, but their nuclei were silent because of the absence of Ki-67 signals and the weak LUC+ signals. These characteristics of the early G1 cells might enhance the development of the reconstructed embryos. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

69 RELATION OF SPATIAL GENE EXPRESSION PATTERNS IN BOVINE EMBRYOS RECONSTRUCTED WITH SOMATIC CELLS TO BLASTOCYST DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 142
Author(s):  
K. Saeki ◽  
T. Tamari ◽  
A. Kasamatsu ◽  
D. Iwamoto ◽  
S. Kameyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Somatic Cells ◽  
Calcium Ionophore ◽  
Gene Expression Patterns ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Reconstructed Embryos ◽  
M Phase ◽  
G1 Cells

Recently, enhanced development to full term was obtained with embryos reconstructed with bovine early G1 cells rather than with G0 cells (Kasinathan et al. 2001 Nat. Biotechnol. 19, 1176-1178; Urakawa et al. 2004 Theriogenology 62, 714-728). However, the reason why donor somatic cells at the early G1 phase are better for embryo reconstruction is unclear. In this study, we investigated the relation of spatial gene expression patterns at the 4- to 8-cell stage to blastocyst development of embryos reconstructed with early G1 cells. Bovine fibroblasts stably transfected with �-act/luc+/IRES/EGFP were used for embryo reconstruction. M phase cells were prepared as described by Urakawa et al. (2004). Early G1 cells were obtained from cultured M phase cells soon after the M phase cells divided. Quiescent cells (cultured in 0.4% serum for 7 days) were used as G0 cells for a control. The cells were electrofused with enucleated bovine oocytes matured in vitro, and activated with a calcium ionophore and cycloheximide. The reconstructed embryos were cultured until 60 hours post fusion (hpf), and zonae pellucidae of 4- to 8-cell embryos were removed by pronase. To determine gene expression, the LUC+ activity (luminescence) in the embryo blastomeres was detected with an imaging photon counter (Hamamatsu Photonics, Hamamatsu City, Shikuoka Prefecture, Japan) for 10 min. The embryos were categorized as being positive, mosaic, or negative depending on whether all, some or no blastomeres were luminescent, respectively. The embryos were cultured in mSOF medium individually until 168 hpf to assess development to the blastocyst stage. Blastocyst development of reconstructed embryos without detection of luminescence was also examined. Experiments were repeated three times, and the data were analyzed with Fisher's PLSD test following ANOVA. At 60 hpf, 75% (74/99) of embryos reconstructed with early G1 cells and 55% (46/83) of embryos with G0 cells developed to 4- to 8-cell stage embryos. The difference is significant (P < 0.05). The percentages of positive, mosaic, and negative embryos with G1 cells were 49, 35 and 16%, and blastocyst rates were 30, 11, and 0%, respectively. With G0 cells, the percentages were 32, 56, and 12%, and the blastocyst rates were 15, 4, and 0%, respectively. More positive embryos were obtained with early G1 cells than with G0 cells (P < 0.05). Blastocyst rates of the positive embryos with early G1 cells were the same as with G0 cells (P > 0.05). Blastocyst development of positive embryos was higher than that of mosaic and negative embryos in early G1 and G0 groups (P < 0.05). Without detection of luminescence, the blastocyst rates from the reconstructed embryos were 43% (35/81) and 16% (20/125) with early G1 and G0 cells, respectively (P < 0.05). These results suggest that the higher developmental capacity of embryos reconstructed with early G1 cells might be related to the appropriate spatial gene expression at the 4- to 8-cell stage. A part of this study was supported by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

scholarly journals Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

1989 ◽  
Vol 9 (5) ◽  
pp. 1940-1945 ◽  
Author(s):  
B Y Tseng ◽  
C E Prussak ◽  
M T Almazan
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Small Subunit ◽  
Mrna Levels ◽  
Proliferating Cells ◽  
Restriction Point ◽  
Serum Stimulation ◽  
G1 Cells

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.

The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression

1996 ◽  
Vol 109 (1) ◽  
pp. 143-153 ◽  
Author(s):  
M. Starborg ◽  
K. Gell ◽  
E. Brundell ◽  
C. Hoog
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Tandem Repeats ◽  
Sequence Motifs ◽  
Mitotic Chromosomes ◽  
Proliferating Cells ◽  
Ki 67 ◽  
Cycle Progression ◽  
Conserved Sequence ◽  
Interphase Cells

We have isolated the murine homologue of the human Ki-67 antigen. The Ki-67 antigen is used as a marker to assess the proliferative capacity of tumour cells; however, its cellular function is not known. The murine Ki-67 cDNA sequence (TSG126) was found to contain 13 tandem repeats, making up more than half of the total protein size. A comparison of this repetitive sequence block to its human counterpart, which contains 16 consecutive repeat units, revealed several conserved sequence motifs, including one motif frequently observed in proteins interacting with DNA. An antiserum developed against the product of the TSG126 cDNA clone identified a protein with an apparent molecular mass of 360 kDa, mainly expressed in proliferating cells. The TSG126 protein begins to accumulate during the late G1 stage of the cell cycle and is first seen as numerous small granules evenly distributed throughout the nucleus. During the S and the G2 phases, larger foci that overlap with the nucleoli and the heterochromatic regions are formed. At the onset of mitosis the TSG126 protein undergoes a dramatic redistribution process and becomes associated with the surface of the condensed chromosomes. The relative absence of the TSG126 protein from G1 interphase cells strongly argues against a model where the association of the TSG126 protein with mitotic chromosomes merely reflects a mechanism for the symmetrical distribution of nucleolar proteins between daughter cells. Instead, the intracellular distribution of the TSG126 protein during the cell cycle suggests that it could have a chromatin-associated function in both interphase and mitotic cells. Microinjection of anti-TSG126 antibodies into proliferating Swiss-3T3 fibroblasts was found to delay cell cycle progression, indicating that the TSG126 protein has an essential nuclear function.

Expression of Cell Cycle Regulatory Proteins and Analysis of Apoptosis in Normal Nasal Mucosa and in Nasal Polyps

2005 ◽  
Vol 19 (6) ◽  
pp. 549-553 ◽  
Author(s):  
Werner Garavello ◽  
Paola Viganò ◽  
Marco Romagnoli ◽  
Lorenza Sordo ◽  
Emilio Berti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nasal Mucosa ◽  
Nasal Polyps ◽  
Normal Mucosa ◽  
Cellular Growth ◽  
Regulatory Proteins ◽  
Biological Behavior ◽  
Proliferating Cells ◽  
Ki 67 ◽  
Sequence Of Events

Background The etiopathogenesis of nasal polyps still is to be clarified. Although hyperplasia is a typical feature of these pathological processes, little attention has been paid to specific aspects of cellular growth in polyps. We have evaluated the expression and localization of some of the regulatory proteins that direct the cell through the specific sequence of events culminating in mitosis or apoptosis in nasal polyps. Methods Twenty samples of nasal polyps and 20 samples of normal nasal mucosa have been analyzed for apoptotic index by detecting the DNA 3’ OH ends deriving from DNA fragmentation. Moreover, they have been evaluated by immunohistochemical staining for expression of Ki-67, cyclins A and B1, p53, p21, p27, murine double minute clone 2, and Bcl-2. Results We have identified a greater proportion of proliferating cells in the lining epithelial cells of the polyps when compared with the normal mucosa as stained with anti–Ki-67 antibodies. An overexpression of p53, MDM2, and Bcl-2 and an increased apoptosis were observed in nasal polyps compared with the normal mucosa, whereas no variation of p27 expression was observed. The p21 and cyclins A and B1 were rarely expressed in both pathological and normal tissue. Conclusion The p53-based control system of cell cycle progression appears to be altered in nasal polyps, potentially leading to an abrogation of the DNA damage checkpoint. Evaluation of the expression of the regulatory proteins that direct the cells throughout their cycle in nasal polyps may allow a better understanding of the biological behavior and clinical outcome of these benign pathological entities.

30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
G. Coppola ◽  
B.-G. Jeon ◽  
B. Alexander ◽  
E. St. John ◽  
D. H. Betts ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Treated Group ◽  
Blastocyst Development ◽  
Fetal Fibroblasts

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

scholarly journals Retinoblastoma 1 (RB1) modulates the proliferation of chicken preadipocytes

2018 ◽  
Author(s):  
Yu-Xiang Wang ◽  
Hai-Xia Wang ◽  
Wei Na ◽  
Fei-Yue Qin ◽  
Zhi-Wei Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phase Transition ◽  
Cell Cycle ◽  
Gene Expression Analysis ◽  
Mammalian Species ◽  
S Phase ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Ki 67

AbstractRetinoblastoma 1 (RB1) has been extensively studied in mammalian species, but its function in avian species is unclear. The objective of this study was to reveal the role of chicken RB1 (Gallus gallus RB1, gRB1) in the proliferation of preadipocytes. In the current study, quantitative real-time PCR analysis showed that the expression levels of gRB1 transiently increased during the proliferation of preadipocytes. The MTT assay showed that gRB1 overexpression suppressed preadipocyte proliferation, and gRB1 interference promoted preadipocyte proliferation. Additionally, cell-cycle analysis indicated that gRB1 may play a crucial role in the G1/S transition. Consistently, gene expression analysis showed that gRB1 knockdown promoted marker of proliferation Ki-67 (MKi67) expression at 96 h (P < 0.05), and that overexpression of gRB1 reduced MKi67 expression at 72 h (P < 0.05). Together, our study demonstrated that gRB1 inhibited preadipocyte proliferation at least in part by inhibiting the G1 to S phase transition.

scholarly journals Cell-to-cell and genome-to-genome variability of adenovirus transcription tuned by the cell cycle

2020 ◽  
Vol 134 (5) ◽  
pp. jcs252544
Author(s):  
Maarit Suomalainen ◽  
Vibhu Prasad ◽  
Abhilash Kannan ◽  
Urs F. Greber
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Single Molecule ◽  
Click Reaction ◽  
Viral Gene Expression ◽  
Early Gene ◽  
Viral Gene ◽  
Viral Genomes ◽  
E1a Gene ◽  
G1 Cells

ABSTRACTIn clonal cultures, not all cells are equally susceptible to virus infection, and the mechanisms underlying this are poorly understood. Here, we developed image-based single-cell measurements to scrutinize the heterogeneity of adenovirus (AdV) infection. AdV delivers, transcribes and replicates a linear double-stranded DNA genome in the nucleus. We measured the abundance of viral transcripts using single-molecule RNA fluorescence in situ hybridization (FISH) and the incoming 5-ethynyl-2′-deoxycytidine (EdC)-tagged viral genomes using a copper(I)-catalyzed azide–alkyne cycloaddition (click) reaction. Surprisingly, expression of the immediate early gene E1A only moderately correlated with the number of viral genomes in the cell nucleus. Intranuclear genome-to-genome heterogeneity was found at the level of viral transcription and, in accordance, individual genomes exhibited heterogeneous replication activity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A gene expression and displayed increased correlation between E1A gene expression and viral genome copy numbers. The combined image-based single-molecule procedures described here are ideally suited to explore the cell-to-cell variability in viral gene expression in a range of different settings, including the innate immune response.

scholarly journals The cell proliferation antigen Ki-67 organises heterochromatin

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Michal Sobecki ◽  
Karim Mrouj ◽  
Alain Camasses ◽  
Nikolaos Parisis ◽  
Emilien Nicolas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Mammalian Cells ◽  
Proliferating Cells ◽  
Ki 67 ◽  
Heterochromatin Formation ◽  
Chromatin Regulators ◽  
Cycle Arrest ◽  
Altered Gene

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.

Ki-67 Labeling Index in Breast Cancer

1989 ◽  
Vol 75 (6) ◽  
pp. 557-562 ◽  
Author(s):  
Sergio Crispino ◽  
Ambrogio Brenna ◽  
Daniela Colombo ◽  
Bajardo Flores ◽  
Silvestro D'Amico ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Monoclonal Antibody ◽  
Menopausal Status ◽  
Mitotic Rate ◽  
Labeling Index ◽  
Nuclear Antigen ◽  
Proliferating Cells ◽  
Cell Cycle Kinetics ◽  
Ki 67

Measurements of cell cycle kinetics have been found to correlate with the clinical course of patients with breast cancer. However, the thymidine labeling index and more rapid methods like flow cytometry remain complicated and costly. We assessed cell proliferation of 67 breast carcinomas by an immunoperoxidase procedure using a monoclonal antibody, Ki-67, which reacts with a nuclear antigen in proliferating cells. The percentage of Ki-67 positive cells ranged from 2% to 70 %. Tumors with high mitotic rate, high nuclear grade, high histologic grade, and negative estrogen receptors had statistically higher Ki-67 labeling rates. We found no significant differences between the Ki-67 labeling rate and other clinical (age at diagnosis, menopausal status) or pathologic (necrosis, fibrosis, vascular invasion, lymphatic invasion, cellular reaction, tumor size, lymph node metastases) features assessed. These results parallel previously reported data, and confirm that this immunohistochemical staining of breast carcinoma by Ki-67 monoclonal antibody can be considered a rapid and convenient method for assessing cell cycle kinetics. However, further studies, evaluating the correlation between Ki-67 labeling rate and prognosis are needed to better define the real usefulness of this analysis in clinical practice.

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