Primordial germ cell-specific expression of eGFP in transgenic chickens.

PR domain zinc finger protein 14 (PRDM14) plays an essential role in the development of primordial germ cells (PGCs) in mice. However, its functions in avian species remain unclear. In the present study, we used CRISPR/Cas9 to edit the PRDM14 locus in chickens in order to demonstrate its importance in development. The eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs to knockout PRDM14 and label PGCs. Chimeric chickens were established by a direct injection of eGFP knocked-in (gene-trapped) PGCs into the blood vessels of Hamburger-Hamilton stages (HH-stages) 13-16 chicken embryos. Gene-trapped chickens were established by crossing a chimeric chicken with a wild-type hen with very high efficiency. Heterozygous gene-trapped chickens grew normally and SSEA-1-positive cells expressed eGFP during HH-stages 13-30. These results indicated the specific expression of eGFP within circulating PGCs and gonadal PGCs. At the blastodermal stage, the ratio of homozygous gene-trapped embryos obtained by crossing heterozygous gene-trapped roosters and hens was almost normal; however, all embryos died soon afterward, suggesting the important roles of PRDM14 in chicken early development.