Genome Editing in Pigs

Area of Science:

• Animal Biotechnology
• Gene Editing Technologies
• Developmental Biology

Background:

• CRISPR/Cas9 has revolutionized the generation of genetically engineered (GE) pigs for disease modeling and xenotransplantation.
• Genome editing in livestock is effectively combined with somatic cell nuclear transfer (SCNT) or microinjection (MI).

Purpose of the Study:

• To provide a detailed laboratory protocol for generating knockout and knock-in porcine somatic cells for SCNT.
• To outline methods for producing knockout pigs via microinjection into fertilized oocytes.

Main Methods:

• Genome editing of porcine somatic cells in vitro for SCNT.
• Microinjection of CRISPR/Cas9 into fertilized porcine oocytes for rapid knockout generation.
• Embryo transfer of manipulated embryos into recipient sows.

Main Results:

• Established protocols for isolation, cultivation, and manipulation of porcine somatic cells for SCNT.
• Detailed procedures for isolation, maturation, and microinjection of porcine oocytes.
• Successful generation of genetically engineered piglets through SCNT and MI.

Conclusions:

• SCNT is advantageous for complex genetic modifications (multi-knockout/knock-in) but is labor-intensive.
• Microinjection offers a faster route to knockout pigs but requires specialized expertise.
• Both SCNT and MI techniques are technically demanding for porcine embryos, necessitating skilled personnel.