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Related Experiment Videos

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

Sharon Harrison1, Andrew Boquest, Christopher Grupen

  • 1BresaGen Limited, Adelaide, South Australia, Australia.

Cloning and Stem Cells
|January 27, 2005
PubMed
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Researchers successfully created alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene knockout pigs using somatic cell nuclear transfer. This advancement in gene editing technology paves the way for further research in genetic modification of livestock.

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.

Related Experiment Videos

  • 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.