An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs

Area of Science:

• Animal Biotechnology
• Gene Editing
• Reproductive Biology

Background:

• Somatic cell nuclear transfer (SCNT) is a key technology for producing genetically modified animals.
• Gene knockout strategies are essential for understanding gene function and developing disease models.
• The alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene is a target for xenotransplantation research.

Purpose of the Study:

• To report the production of alpha1,3-GT gene knockout pigs using established SCNT and gene targeting methods.
• To evaluate the efficiency of gene targeting in porcine fetal fibroblasts.
• To assess the efficiency of SCNT for producing gene knockout piglets.

Main Methods:

• Porcine fetal fibroblasts were targeted to knock out the alpha1,3-GT gene using a nonisogenic promoterless construct.
• Gene-targeted clones were identified using long-range PCR.
• Nuclear transfer was performed using a fusion before activation protocol with adult oocytes.
• Embryos were transferred to synchronized recipients, and pregnancies were monitored.

Main Results:

• A targeting efficiency of 6.5% was achieved, identifying seven alpha1,3-GT gene knockout cell clones.
• Three cell clones were used for SCNT, resulting in the birth of 18 live piglets from four recipients.
• All resulting pigs were confirmed to be alpha1,3-GT gene knockouts via PCR and Southern blot analysis.
• The SCNT protocol achieved a 1.9% efficiency (embryos transferred/piglets born).

Conclusions:

• The study successfully produced alpha1,3-GT gene knockout pigs using SCNT and a nonisogenic promoterless construct approach.
• The findings demonstrate the feasibility of efficient gene targeting and SCNT for generating genetically modified pigs.
• This work provides a foundation for further applications of gene editing in porcine models.