31 CONSTRUCTION OF A TARGETING VECTOR SPECIFIC FOR THE BOVINE BETA-CASEIN GENE
Notice bibliographique
Résumé
Although using livestock as animal bioreactors is a powerful tool to produce valuable therapeutic proteins in the milk, there are still some limitations to the technology such as a low frequency of transgenesis and a low expression of the transgenes by random integration. In this study, we constructed gene-targeting vectors for a mammary gland-specific gene and then obtained two homologous recombinant cell clones after transfection of the vectors into bovine somatic cells. Two targeting vector cassettes, BCKI I and BCKI II, which have homology regions for a bovine beta-casein gene, were constructed. The beta-casein gene is expressed only in the mammary gland and is the most abundant milk protein in the cow. The targeting sequence lengths of the BCKI I and BCKI II vector cassettes were 13.1 kb and 9.1 kb, respectively, and contained different long arm lengths. A neo gene was inserted into the vectors as a selection marker, and a few restriction enzyme sites were made in front of the neo gene. The human thrombopoietin (TPO) gene was inserted into the restriction enzyme sites of the vector cassettes, named BCTPOKI I and BCTPOKI II vectors. The BCTPOKI I and BCTPOKI II vectors were transfected into bovine embryonic fibroblasts (bEF) and ear skin fibroblasts (bESF) using Lipofectamine™ 2000 reagent (Invitrogen, Seoul, South Korea). In order to determine the highest transfection efficiency, a variety of factors such as DNA concentration, lipid volume, and exposure time to DNA-liposome complexes based on the manufacturers' guideline, was optimized. The 2:1 and 1:2 ratios of DNA (µg) to transfection reagent (µL) were efficient for bEF and bESF, respectively, under overnight exposure to DNA-Lipofectamine™ 2000 reagent. Seventeen percent (51/304) of bESF clones and 6% (9/149) of bEF clones were normally expanded into passage 8. PCR and Southern blotting indicated that 6.3% (2/32) of the clones carrying with BCTPOKI II vectors was homologously targeted at the beta-casein gene. However, none (0/60) of the clones carrying BCTPOKI I was targeted. Additionally, both of the targeted clones were from bESF. When the targeted cells were transferred into enucleated oocytes and cultured, 83% (43/52) of the cloned embryos were transgenic. Thus, we found that homologous recombinant events using gene-targeting vectors might be dependent on cell types, vector sizes, and transfection procedures. In conclusion, mammary gland-specific gene-targeting vectors coupled with somatic cell nuclear transfer technology will be very useful for developing animal bioreactors that produce therapeutic proteins in milk.
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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 ».