An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs.
Cloning Stem Cells
; 6(4): 327-31, 2004.
Article
in En
| MEDLINE
| ID: mdl-15671659
ABSTRACT
We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) 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. alpha1,3-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.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Oocytes
/
Swine
/
Gene Deletion
/
Nuclear Transfer Techniques
/
Fibroblasts
/
Galactosyltransferases
Limits:
Animals
/
Pregnancy
Language:
En
Journal:
Cloning Stem Cells
Journal subject:
GENETICA
Year:
2004
Document type:
Article
Affiliation country:
Australia