A Comparative Study of Transfection Techniques for Genetic Modification in Chicken Primordial Germ Cells

Avian species possess a distinct reproductive system that differs fundamentally from mammals, resulting in relatively slower progress in transgenic poultry research compared to mammalian models. Primordial germ cells (PGCs), as the precursors to functional gametes, provide a promising avenue for the utilization of genetic resources and the application of transgenesis in chickens. However, the transfection of chicken PGCs remains challenging due to the low efficiency of conventional methods. This study systematically compared the transient transfection efficiency of chicken PGCs using lipofection, electroporation, adenovirus (ADV), and adeno-associated virus (AAV) to identify the optimal genes delivery approach. Lipofection exhibited less than 5% efficiency at 24 h, whereas electroporation significantly increased transfection efficiency but reduced cell viability at 48 h. Adenoviral vectors achieved the highest delivery efficiency 8-10 fold greater than lipofection, surpassing both lipid-mediated transfection and electroporation, while adeno-associated viruses serotypes showed minimal infectivity in PGCs. Furthermore, ADV-transduced PGCs retained normal migratory capability after transplantation into 2.5-day chicken embryos, confirming that adenoviral infection did not impair their biological functionality. Collectively, our findings established adenoviral delivery as a robust and efficient method for gene transfer in chicken PGCs, enabling precise genome engineering for applications in transgenic avians production and the conservation of endangered poultry genetic resources.