Germ cell-directed exosomal delivery enables in vivo genetic modification in birds

Avian embryos present substantial barriers to early-stage genetic manipulation due to the rigid eggshell and opaque yolk, underscoring the need for alternative in vivo germline delivery systems. Extracellular vesicles (EVs), particularly exosomes, are biocompatible nanoscale carriers of nucleic acids, yet their potential for germline targeting in birds has not been investigated. Here, we isolated exosomes from long-term cultured chicken primordial germ cells (PGCs) and characterized their morphology, size distribution, and exosomal marker expression. PGC-derived exosomes efficiently delivered plasmid DNA into primary PGCs with minimal cytotoxicity. In vivo microinjection of plasmid-loaded exosomes into early chicken and quail embryos induced robust transgene expression within developing embryonic gonads, demonstrating their ability to traverse embryonic barriers and access germline niches. Vector-derived sequences persisted in embryonic and post-hatch gonads, and GFP-positive germ cells were detected in the testes of sexually mature G₀ quails, supporting preferential germline uptake and expression. Collectively, these findings establish PGC-derived exosomes as promising germline-directed nanocarriers for in vivo gene delivery in birds, with potential applications in avian genome engineering and germline modification.