Meng Lu, Zhu Zimo, Xue Man, Wang Feiyi, Ma Yuxiao, Zhu Guiyu
Shandong Provincial Key Laboratory for Livestock Germplasm Innovation & Utilization, College of Animal Science, Shandong Agricultural University, Taian, China.
Mol Biotechnol. 2025 Sep 5. doi: 10.1007/s12033-025-01501-w.
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.
鸟类拥有独特的生殖系统，与哺乳动物的生殖系统存在根本差异，这导致转基因家禽研究与哺乳动物模型相比进展相对较慢。原始生殖细胞（PGCs）作为功能性配子的前体，为鸡遗传资源的利用和转基因应用提供了一条有前景的途径。然而，由于传统方法效率低下，鸡PGCs的转染仍然具有挑战性。本研究系统地比较了脂质转染、电穿孔、腺病毒（ADV）和腺相关病毒（AAV）对鸡PGCs的瞬时转染效率，以确定最佳的基因递送方法。脂质转染在24小时时效率低于5%，而电穿孔显著提高了转染效率，但在48小时时降低了细胞活力。腺病毒载体实现了最高的递送效率，比脂质转染高8至10倍，超过了脂质介导的转染和电穿孔，而腺相关病毒血清型在PGCs中的感染性最小。此外，ADV转导的PGCs在移植到2.5天的鸡胚后保留了正常的迁移能力，证实腺病毒感染并未损害其生物学功能。总的来说，我们的研究结果确立了腺病毒递送作为一种强大而有效的鸡PGCs基因转移方法，能够实现精确的基因组工程，用于转基因禽类生产和濒危家禽遗传资源的保护。
