Establishing an induced infertile chicken line for efficient germline transmission of exogenous PGCs

Primordial germ cells (PGCs) are the stem-cell population of adult animal gametes, which develop into sperm or eggs. It can be propagated in vitro and injected into the host chicken for genome editing to obtain germline chimeric chicken. However, it has the limitation that the host embryo contains endogenous PGCs, which raises complications, resultantly donor PGCs fail to compete, and transmission efficiency reduced. Therefore, to increase the transmission efficiency, we generated a novel sterile chicken with the inducible elimination of endogenous PGCs in the host. This is the first study that applied the herpes simplex virus thymidine kinase (HSV-TK) cell ablation system in avian. CRISPR/Cas9-mediated homology-directed repair was performed to localize the HSV-TK suicide gene to the last exon of the deleted in azoospermia-like (DAZL) gene, and ganciclovir (GCV) was added to induce the apoptosis in the germ cells of the host embryo. The sterilized host embryo introduced genome-edited PGCs to produce chimeric chicken carrying exogenous germ cells only. It was observed that the germline transmission efficiency was 100% achieved, and the obtained chicks were purely from donor breeds. The technologies established in the current study have important applications in germplasm conservation and gene editing in chicken.