43 DNA SYNTHESIS, PREIMPLANTATION DEVELOPMENT AND Oct-4 EXPRESSION OF BOVINE CLONES RECONSTRUCTED WITH OOCYTES PREACTIVATED OR ENUCLEATED AFTER SPINDLE DISASSEMBLY
Notice bibliographique
Résumé
Enucleation procedures applied in mammalian cloning remove not only the oocyte's chromosomes but presumably also the spindle-associated factors. If these factors are beneficial for reprogramming, alternative protocols that limit enucleation of factors in addition to the chromosomes may improve cloning efficiency. In this study, we evaluated the enucleation in combination with various activation protocols on clone development and gene expression. Clones produced by nuclear transfer into pre-activated bovine oocytes rather than non-activated oocytes can develop in vitro (Kurosaka et al. 2002 Biol. Reprod. 67, 643–647; Tani et al. 2003 Biol. Reprod. 69, 1890–1894). We produced bovine clones using four different nuclear transfer protocols and, in clones of all groups, examined timing of DNA synthesis in the first cell cycle, pre-implantation development, and gene expression at the blastocyst stage. Protocols applied were: (A) donor cells were fused with non-activated oocytes; (B) donor cells were fused with oocytes at 2 hours after activation with ethanol (7%, 7 min); (C) oocytes were enucleated after spindle disassembly with nocodazole treatment (0.3 µg/mL, 30 min) and donor cells were fused with non-activated oocytes; and (D) oocytes were enucleated after spindle disassembly and donor cells were fused with oocytes at 2 h after activation. Fused couplets in all treatment groups were treated with 10 µg/mL cycloheximide for 6 h, and cultured in vitro in SOF supplemented with fetal bovine serum at 39°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The onset of DNA synthesis was determined by an immunofluorescence assay of 5-bromo-deoxyuridine incorporation at 6 and 9 h post-fusion (hpf). Oct-4 mRNA distribution in clone blastocysts was examined by whole mount in situ hybridization using a bovine Oct-4-specific antisense riboprobe. Data were statistically analyzed with Student's t-test. In the majority of clones DNA synthesis had not commenced 6 hpf but had initiated 9 hpf. Although the cell cycle of activated oocytes (protocols B and D) was 2 hours advanced compared to non-activated oocytes (protocols A and C), clones produced by all protocols had a similar onset of DNA synthesis at 6 to 9 h post-fusion. Developmental rates to the blastocyst stage of clones were not significantly different between the four protocols (48.5%–57.7%, P < 0.05). Oct-4 distribution in clones produced by all four protocols was not different from that of IVF embryos used as a control in that Oct-4 mRNA signal was typically restricted to the ICM (87.0%–100.0%, P < 0.05). We conclude that in bovine clones produced in this study, nocodazole-treated enucleation and activation status of recipient oocyte did not influence the pre-implantation development and spatial pattern of Oct-4 expression. This work was supported by the Lalor Foundation.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».