Inserting random and site-specific changes into the genome of chickens

Area of Science:

• Genomics and Molecular Biology
• Avian Biotechnology
• Transgenesis

Background:

• Chicken genome modification has progressed from random viral vector insertions to precise gene editing and large transgene integration.
• Primordial germ cells (PGCs) are key to germline modification, retaining functional gamete potential after in vitro culture.
• Stable germline modification rates vary significantly between targeted and nontargeted insertion methods.

Purpose of the Study:

• To detail advancements in chicken genome engineering using primordial germ cells (PGCs).
• To outline methods for generating genetically modified chickens with specific traits.
• To demonstrate the application of these techniques for creating disease models and producing therapeutic proteins.

Main Methods:

• Culturing and genetically modifying PGCs in vitro.
• Injecting modified PGCs into early-stage chicken embryos (Hamburger and Hamilton stages 13-15).
• Utilizing Green Fluorescent Protein (GFP) for tracking PGC colonization and flow cytometry/PCR for assessing genetic contribution.
• Breeding chimeric chickens to obtain G1 heterozygote offspring.

Main Results:

• Successful generation of chickens with targeted gene knockouts (e.g., immunoglobulin genes) and transgene insertions.
• Demonstrated production of chickens with human antibody V regions and chickens expressing GFP.
• Established protocols for creating chickens expressing Cre recombinase for conditional gene deletion.

Conclusions:

• The described PGC-based genome modification protocol is effective for creating diverse genetically engineered chicken models.
• These advancements facilitate research into avian immunology, disease resistance, and the production of valuable biomolecules.
• The methodology provides a robust platform for future genetic studies and applications in poultry.