Abstract
We conducted the present study to develop a simplified method of isolating gonadal germ cells (GGCs) from embryonic gonads to facilitate the production of germline chimeras in avians. Developing gonads recovered from 7-day-old chick embryos were incubated for 30 minutes to 24 hours at 37.8°C in phosphate-buffered saline without Ca2＋ and Mg2＋ (PBS[－]). GGCs began to be released from gonads after 30 minutes of incubation; the number of GGCs release increased until 12 hours of incubation. The purity of GGCs (the number of GGCs released divided by the total number of cells released) was approximately 50% for the initial 1.5 hours of incubation and decreased thereafter. We stained 50 GGCs in PBS[－] with PHK26 fluorescent dye and injected them into the bloodstream of a 2-day-old chick embryo. GGCs exhibiting fluorescence were detected in the gonads of recipient embryos at 5 days after transfer. These results indicate that hig-purity, highly viable GGCs can be isolated from chick embryos simply by incubating the developing gonads in PBS[－].
