Generation of chimeric chickens resistant to infection by avian leukemia virus subgroups A, B, D, E, and K through primordial germ cells

Avian leukosis virus (ALVs), an oncogenic retrovirus, induces immunosuppression and various tumors in chickens, posing a severe global threat to poultry health and causing substantial economic losses. Natural resistance alleles, notably tva^r2 and tvb^r3, confer genetic resistance to specific ALV subgroups (ALV-A, -K, and ALV-B, -D, -E, respectively). This study aimed to exploit this resistance by creating resistant chicken lines via primordial germ cells (PGCs)-mediated chimera. We first genotyped the tva^r2 and tvb^r3 loci in Qingyuan Partridge Chickens (QYPCs) and Zhanjiang Yellow Chickens (ZJYCs). Through selective mating, we generated homozygous tva^r2/r2 and tvb^r3/r3 QYPC embryos as PGC donors, and homozygous susceptible tva^s/s and tvb^s/s ZJYC embryos as recipients. Donor PGCs harvested from QYPC embryos were successfully cultured in vitro, as confirmed by the expression of germ cell-specific markers (DDX4, c-Kit, PRDM14) and scanning electron microscopy revealing characteristic microvilli. These PGCs were microinjected into recipient embryos, producing viable chimeric chickens. Genotyping confirmed the successful integration of the resistant donor-derived PGCs. The germline transmission efficiency reached 35.7%. Our findings demonstrate that PGC-mediated germline transmission provides an efficient strategy for rapidly establishing ALV-resistant poultry flocks, offering a potential strategy for establishing ALV-resistant poultry flocks by introgressing resistance alleles.