28 PREGNANCIES RESULTED FROM GOAT NT EMBRYOS PRODUCED BY FUSING COUPLETS IN THE PRESENCE OF LECTIN
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Notice bibliographique
Résumé
The procedure of nuclear transfer (NT) using somatic cells remains inefficient partly due to low fusion rates between donor cells and recipient ooplasm. Lectin is a glycoprotein which specifically binds to carbohydrates to induce a tight contact of membrane to membrane (Booth et al., 2001 Cloning and Stem Cells, 3, 139–159). The purpose of this study was to examine the fusion rates and developmental competence of NT embryos following pre-incubation of couplets in medium containing lectin prior to electrical pulsing. Oocytes were collected by laparoscopic ovum pick-up from hormonally primed goats or by aspiration from culled goat ovaries, and cultured for maturation at 38.5°C, 5% CO2. At approximately 24 h after the onset of IVM, the cumulus cells were stripped off by brief vortexing in medium containing 0.2% hyaluronidase. Oocytes with first polar bodies were selected for NT. Successful enucleation was confirmed by the absence of MII chromosomes in ooplasm by means of brief exposure of the Hoechst 33342-stained oocytes to UV light. Three cumulus-granulosa cell lines from transgenic goats were used as donor cells. They were cultured to confluency in DMEM + 20% FCS for 6 days prior to NT. Individual donor cells were transferred into the perivitelline space of the enucleated oocytes. Couplets were incubated for 15 minutes in TCM199 + 10% FCS containing 75 or 150 µg mL-1 lectin (L-9132 Sigma, St.Louis, MO, USA) prior to being subjected to electrical pulsing (lectin treatment) with one DC pulse at 2.4 kV/cm (1st pulsing). The fusion rate was determined 40–60 minutes after the 1st electric pulsing. Non-fused couplets were exposed to a 2nd pulsing. Approximately 30 minutes later, non-fused couplets were exposed to a 3rd pulsing. Couplets without the lectin treatment served as controls. Reconstructed embryos were activated with 5 µM ionomycin followed by 5 h of incubation in 10 µg mL-1 cycloheximide with 7.5 µg mL-1 cytochalasin B. A group of 10 to 13 embryos was transferred into a recipient after 12 to 14 h of culture in G1.3. Statistical analysis was performed using the ?2 analysis. Results are shown in the following table. This study demonstrated that fusion rate could be improved by pre-incubating couplets in the medium containing 150 µg mL-1 lectin prior to electrical pulsing and the embryos derived from the lectin treatment could establish the early pregnancies.
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Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,002 | 0,002 |
| 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 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
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 découle