| Autor: |
Harrison, Sharon, Boquest, Andrew, Grupen, Christopher, Faast, Renate, Guildolin, Angelo, Giannakis, Christopher, Crocker, Lesley, McIlfatrick, Stephen, Ashman, Rodney, Wengle, James, Lyons, Ian, Tolstoshev, Paul, Cowan, Peter, Robins, Allan, O'Connell, Philip, D'Apice, Anthony J.F., Nottle, Mark |
| Zdroj: |
Cloning and Stem Cells (formerly Cloning: Science and Policy); Winter 2004, Vol. 6 Issue: 4 p327-331, 5p |
| Abstrakt: |
We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the α(1,3)-galactosyltransferase (α1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of α1,3-GT gene knockout pigs using these procedures. Seven α1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neoR) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro–matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. α1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth. |
| Databáze: |
Supplemental Index |
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