A Step-by-Step Procedure for Producing Germline Chimera in Chicken <em>via</em> Primordial Germ Cell Transplantation

The generation of germline chimeras in chickens via transplantation of primordial germ cells (PGCs) provides a robust and reproducible platform for avian transgenesis, genome editing, and species conservation. Traditional approaches using blastodermal cells from EGK stage X embryos are limited by low germline transmission efficiency due to early cell lineage segregation. In contrast, PGC-mediated strategies exploit the intrinsic germline competency of these unipotent cells, enabling reliable incorporation into recipient gonads. Here, we present a stepwise protocol for isolating PGCs from HH stage 26-28 embryos, maintaining and expanding them in vitro, and transplanting them into the dorsal aorta of HH stage 14-17 recipient embryos. Short-term engraftment is monitored using fluorescent markers such as PKH26, while long-term germline contribution is confirmed by PCR analysis of recipient gonads. This approach ensures consistent generation of germline chimeras while preserving the viability and functional competence of donor PGCs. Additionally, the method is compatible with genome editing tools such as CRISPR/Cas9 and transposon-based vectors, enabling the production of transgenic and gene-edited avian models. Beyond basic research, this strategy supports conservation efforts through interspecies germline transmission and genetic resource preservation. Overall, this protocol provides a comprehensive and reliable framework for manipulating the avian germline, offering a versatile platform for both fundamental and applied studies in poultry biotechnology.