27 SODIUM CHLORIDE TREATMENT OF CELL MEMBRANE-PERMEABILIZED NUCLEAR DONOR CELLS FACILITATES THE DISPLACEMENT OF THE SOMATIC HISTONE H1 AND HMG-17 PROTEINS IN RECONSTRUCTED PORCINE EMBRYOS
Bibliographic record
Abstract
Developmental efficiency of somatic cell-reconstructed embryos depends on extensive remodeling of chromatin structural components. Due to their importance for maintaining the high-order chromatin structure and controlling DNA functions, including replication, transcription, repair, and recombination, histones and other chromatin-binding proteins represent leading choice markers to investigate nuclear remodeling in reconstructed embryos. The main objective of this study was to investigate whether or not the exposure of cell membrane permeabilized nuclear donor cells to sodium chloride (salt-extraction) would facilitate the displacement of chromatin-binding proteins in reconstructed porcine embryos. Both linker histone H1 (H1) and high-mobility group (HMG) proteins are known to affect gene expression through the modulation of the high-order chromatin structure. Standard methods of oocyte enucleation and electrofusion were applied for embryo reconstruction using in vitro-matured oocytes and control or salt-extracted fetal fibroblast cells. For salt-extraction, confluent cell cultures were washed with Ca2+/Mg2+-free Hank's balanced salt solution (HBSS); cells were permeabilized by incubation with 1 µg/mL of streptolysin O at 37°C for 30 min in HBSS, and then maintained in Tris-NaCl buffer (10 mM Tris-HCl, 0.5 mM MgCl2, 0.7 M NaCl, 1 M sucrose) for 5 min. After salt-extraction, cells were rinsed and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 2 mM CaCl2 for 1 h at 37.5°C for membrane resealing prior to nuclear transfer. Reconstructed embryos were activated using ionomycin (15 µM/5 min) and strontium chloride (Sr2+; 10 mM/4 h), and then cultured in PZM-3 medium. Immunostaining for H1 and HMG-17 was performed in nuclear donor cells and embryos at different stages after reconstruction. The time required for H1 displacement in transplanted nuclei was reduced by the salt-extraction treatment (Table 1). Salt-extracted cells showed a stronger HMG-17 cytoplasmic signal compared to control cells. The proportion of HMG-17-positive reconstructed embryos at 1, 3, and 6 h was 54 vs. 19, 57 vs. 44, and 75 vs. 62, for control and salt-extracted cells, respectively. These data suggest that salt-extraction prior to nuclear transplantation enhances the remodeling of chromatin structure in embryos reconstructed with somatic cell nuclei. Table 1. Proportion (n) of H1-positive stained embryos after different times from parthenogenetic activation (PA) and nuclear transfer using control (NT-control) or salt-extracted (NT-extracted) cells This work was supported by a NSERC Discovery Grant to VB.
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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) | 0.003 | 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".