40 CHARACTERIZATION OF EARLY G1 CELLS AS NUCLEAR DONORS FOR SOMATIC CELL CLONING IN CATTLE
Bibliographic record
Abstract
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 ß-actin/firefly luciferase fusion gene (ß-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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".