254 IDENTIFICATION OF OOCYTE SPECIFIC GENES USING SSH ANDMICROARRAY ANALYSIS
Notice bibliographique
Résumé
The main objective of this project was to isolate and identify important genes specifically expressed in oocytes. These genes are characterized and may uncover the molecular mechanisms related to the unique functions found in the oocyte. Total RNA (1 µg) from denuded germinal vesicle-stage (GV) oocytes and somatic tissues were used for cDNA production. The mRNAs were reverse-transcribed and the cDNAs were amplified using the Smart cDNA amplification kit (Clontech, BD Biosciences, San Jose, CA, USA). SSH was performed with the PCR-Select cDNA Subtraction Kit (Clontech). Briefly, pools consisted of bovine oocytes for the tester and bovine somatic tissues for the driver. The same procedure was repeated with mouse and xenopus tissues. All of the 3500 subtracted PCR products generated by SSH were cloned, PCR amplified and sequenced. The resulting sequences were compared against the GenBank database using online computer BLASTn program. For microarray analysis, purified PCR products were spotted on GAPS II glass slides (Corning, Corning, NY, USA) using a VersArray Chip WriterPro robot (BioRad, Hercules, CA, USA). Forward- and reverse-subtracted PCR products were used as probes labeled with Cy-3 and Cy-5 dyes (Amersham, Piscataway, NJ, USA) using the Amino Allyle cDNA Labeling Kit (Ambion, Austin, TX, USA). Slides were scanned and analyzed using the ChipReader and ArrayPro Analyser software (Media Cybernetics, Carlsbad, CA, USA). Detection of the oocyte-specific zona pellucida (ZP), growth differentiation factor-9 (GDF-9), bone morphogenetic protein 15 (BMP15), H1 histone family, member O, oocyte-specific (H1oo), and cyclin B1 transcript in the oocyte subtracted library increased confidence in the validity of the subtraction procedure. All of these transcripts account for 12 % of the clones for the mouse subtracted oocyte library. Microarray analysis performed with the mouse arrays revealed that 33% (382) of the clones were differentially expressed (ratio > 10) in the oocyte for the mouse library. Of the 139 clones (12%) that seemed to be present only in the oocyte, 65 were found to be expressed in both the bovine and the xenopus subtracted oocyte library. The most interesting clones to date are #1518, a gene associated with pluripotency;; #1776, a cDNA associated with stem cells in mouse, and #1906, a protein that interacts with chromatin. Since they are conserved across species, the chances that their function is important are quite high. A validation step with RT-PCR analysis will need to be performed on genes identified as oocyte-specific, and the functionality of these genes will be determined with the RNAi technique. WEB page http://www.begc.crbr.ulaval.ca/ (Supported by NSERC).
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 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 | 0,001 |
| 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 ».