Expression of GFP Gene in Cultured PGCs Isolated from Embryonic Blood and Incorporation into Gonads of Recipient Embryos

Abstract

The present study was conducted to develop a technique for generating primordial germ cells expressing GFP gene and introducing them into gonads of recipient embryos. Primordial germ cells isolated from embryonic blood were cultured on feeder cells for more than 40 days. They proliferated, occasionally formed cell colonies, and showed the characteristics of germline cells as detected by anti-CVH antibody. The cultured PGCs were transferred to the stage X blastoderm, bloodstream of stages 14-15 embryos, and the coelomic epithelium of stages 17-19 embryos, and examined to determine whether they could migrate to the gonads of recipient embryos. As a result, they successfully entered the gonads of recipient embryos when they were transferred to the coelomic epithelium, although they failed to migrate to the gonads of recipient embryos when they were transferred to the stage X blastoderm or bloodstream. The cultured PGCs were then transfected with GFP gene by nucleofection and selected for PGCs expressing GFP gene in the presence of G418. Cultured PGCs expressing GFP gene proliferated slowly, forming cell colonies, and successfully entering the gonads by transferring into the coelomic epithelium of recipient embryos. Those results suggest that gene transfer into the chicken germline is possible via cultured PGCs, and that the PGC culture system thus holds enormous possibilities for avian embryo manipulation.