65 PRODUCTION OF PORCINE NUCLEAR TRANSFER EMBRYOS USING FETAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS
Bibliographic record
Abstract
One of the most important factors that determine the developmental potential of mammalian cloned embryos is the structuro-functional quality of nuclear donor cells. Biochemical changes that are some of the earliest symptoms of apoptosis signal transduction are not reflected in the morphological features of somatic cells. Therefore, an appropriate system of cell selection would enable the sorting of donor nuclei with high morphological and biochemical susceptibility to somatic cloning. The aim of our study was to examine the in vitro developmental competencies of porcine nuclear transfer (NT) embryos reconstructed with fetal fibroblast cells that had been analyzed for apoptosis by live-fluorescent labelling. Frozen/thawed fetal fibroblast cells, which had been in vitro-cultured to a confluent state, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, a single cell suspension of nuclear donor cells was subjected to dyeing with live-DNA green fluorochrome YO-PRO-1. The recipient cells were in vitro-matured oocytes. Maternal chromosomes were removed by a chemically assisted microsurgical technique. Then, single nuclear donor cells were inserted into the perivitelline space of enucleated oocytes. Fibroblast cell-ooplast couplets were simultaneously fused and activated with two consecutive DC pulses of 1.2 kV/cm for 60 µs. Reconstructed embryos were in vitro cultured in 50-µL drops of NCSU-23 medium supplemented with 0.4% BSA-V for 6 to 7 days at 38.5°C in a humidified atmosphere of 5% CO2 and 95% air. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of approximately 50 different random samples collected from the population of fetal fibroblast cells, that had been labelled with YO-PRO-1 dye, it was found that a relatively high proportion of donor cells revealed ultrastructural apoptotic changes. The percentage of late apoptotic cells with advanced morphological transformations was about 40% of the total pool of the fibroblast cells. A total of 262/270 (97.0%) enucleated oocytes were subjected to reconstruction and 141/262 (53.8%) were successfully fused with non-apoptotic nuclear donor cells. Following the simultaneous fusion/activation protocol, reconstituted oocytes were selected for in vitro culture. Out of 262, 133 (50.8%) cultured NT embryos cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/133 (36.0%) and 10/133 (7.5%), respectively. In conclusion, morphology is a sufficient selection factor for detection of apoptosis in the cultured (confluent) fetal fibroblast cells to be used for cloning. Moreover, it was found that YO-PRO-1 fluorochrome may be not able to detect the early phases of apoptosis, because only the morphologically abnormal cells emitted the YO-PRO-1-derived fluorescence. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-KBN-084/P06/2002/4.2 from years 2003 to 2005.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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) | 0.002 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".