Characterization of developmental potential in isolated medaka blastomeres and cultured embryonic cells

Cultured mouse embryonic stem (ES) cells have been successfully used to produce transgenic animals. However, in vitro culture of fish ES cells capable of forming germ-line chimeras are not yet available. In an attempt to establish a method for the production of transgenic fish using cultured ES cells from a freshwater fish medaka (Oryzias latipes), we analyzed the developmental potential of the isolated blastomeres and cultured embryonic cells by transplanting them into blastula-stage embryos. Isolated blastomeres were pluripotent, and they participated to form fish chimeras when transplanted into the recipient embryos. Differentiation of the transplanted cells in chimeric fish could be detected by the appearance of the donor phenotype ic. body pigmentation or among their progeny. We report here that the frequency of germ-line transmission of the transplanted cells (36%) was higher than the frequency of body pigmentation (23.9%). The results indicated the possibility of the production of transgenic fish from ES cells. Isolated blastomeres were then cultured with the feeder cell layer. The feeder cells were derived from 5-day-old medaka embryos. Depending on culture conditions, the embryonic cells either formed ES-like colonies or differentiated in vitro. In some cases, they formed structures similar to the posterior body trunk of the fish. When the embryonic cells were cultured for two weeks and transplanted into the recipient embryos, however, they did not show pigmentation or germ-line transmission in the transplants. The results indicated that the embryonic cells had lost their potential to form chimeric fish after prolonged incubation.