72 EFFECTS OF GENE EXPRESSION IN BOVINE EMBRYOS RECONSTRUCTED WITH FIBROBLASTS TRANSFECTED WITH LUCIFERASE GENE ON THE SUBSEQUENT DEVELOPMENT
Notice bibliographique
Résumé
During embryo development, embryonic gene activation (EGA) is one of the first critical events. Inappropriate EGA results in failure of further development. We have reported that gene expression in bovine embryos reconstructed with fibroblasts begins at 48 hours postfusion (hpf) and reaches a maximum level at 60 hpf as detected by their bioluminescence following injection of chicken ß-actin/firefly luciferase fusion gene (ß-act/luc+) into their nuclei (Saeki et al., 2001 Theriogenology 55, 289). In the present study, effects of gene expression in embryos reconstructed with bovine fibroblasts transfected with luciferase gene on their subsequent development to the blastocyst stage were examined. Cultured bovine fibroblasts taken from an ear of a female calf were transfected with plasmid containing ß-act/luc+/IRES/EGFP and neor using GeneJammer (StrataGene, La Jolla, CA, USA). Neomycin-resistant cells were selected by culturing with G418. Then, EGFP-positive colonies were further selected under fluorescence microscopy to obtain stably transfected cells. The transfected cells were cultured for several passages. Growing (50 to 60% confluence, GCs) and serum-starved cells (SCs) were 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 calcium ionophore and cycloheximide. Luminescence in the embryos was detected with an imaging photon counter at 0 and 60 hpf. Luminescence-positive (P) and -negative (N) embryos were cultured separately at each detection time. Embryos were cultured until 168 hpf, and examined for cleavage and blastocyst development. Experiments were repeated 3 times, and totals of 91 and 123 embryos were reconstructed with GCs and SCs, respectively. Data were analyzed with Fisher’s PLSD test following ANOVA by Stat View software (Ver. 5.0). At 0 hpf, luminescence was detected in 55 and 4% of embryos reconstructed with GCs and SCs, respectively. At 60 hpf, luminescence was detected in 47 and 28% of P and N embryos with GCs, and 17 and 40% of P and N embryos with SCs at 0 hpf, respectively. Cleavage rates were not different among groups (P > 0.05). Blastocysts were obtained only from the groups of embryos that were N at 0 hpf and P at 60 hpf (8% with GCs and 17% with SCs). No embryos in the other groups developed to the blastocyst stage. These results suggest that appropriate gene expression in embryos reconstructed with somatic cells is important for their subsequent development and that detecting the reporter gene expression can be used for selection of viable cloned embryos.
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
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 | 0,001 | 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 | 0,000 |
| 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 découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, 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 ».