62 RELATION OF INTENSITY OF GENE EXPRESSION IN BOVINE RECONSTRUCTED EMBRYOS TO SUBSEQUENT DEVELOPMENT
Bibliographic record
Abstract
During embryo development, embryonic gene activation (EGA) is the first critical event. We previously showed that EGA is also critical for further development in somatic cell-cloned embryos (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). To show this, we reconstructed bovine embryos with bovine somatic cells transfected with chicken ß-actin/firefly luciferase fusion gene (ß-act/luc+) and showed that only luminescent embryos at 60 hours post-fusion (hpf) developed to the blastocyst stage. In this study, we examined the relation between the intensity of expression of the same reporter gene in embryos reconstructed with bovine ß-act/luc+ fibroblasts and their subsequent development to the blastocyst stage. Bovine fibroblasts were transfected with ß-act/luc+ as described earlier (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). The stably transfected and cloned cells were cultured for several passages. The cells were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20 h post maturation. Enucleated oocytes were electrofused with the cells, and activated with a calcium ionophore and cycloheximide. The LUC+ signal (luminescence) in the embryos was detected in medium containing 500 µg mL-1 luciferin with an imaging photon counter (ARGUS 50, Hamamatsu, Japan) for 30 consecutive min at 60 hpf. The intensity of luminescence in embryos (4- to 8-cell stage) was graded as being strong (>10 × 104 pixels/embryo), intermediate (5 to 10 × 104 pixels/embryo), weak (<5 × 104 pixels/embryo), or absent. The embryos were cultured separately until 168 hpf, and examined for blastocyst development. Experiments were repeated four times, and the data were analyzed with Fisher's PLSD test following ANOVA by Stat View software (Ver. 5.0; abacus Concepts, Berkeley, CA, USA). Of 125 embryos that were reconstructed, 74 (59%) developed to the 4- to 8-cell stage at 60 hpf. The luminescence was strong in 29 (39%) of the embryos, intermediate in 12 (16%), weak in 19 (26%), and absent in 14 (19%). Blastocysts were obtained from a group of embryos that exhibited strong luminescence (10/29, 34%), but none of the embryos from the other groups developed to blastocysts. These results suggest that active gene expression in embryos reconstructed with somatic cells is important for their subsequent development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Ministry of Education, Culture, Sports, Science, and Technology, 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".