I-18: Avian Chimeras and Germ Cell Migration

Background: In avian species, the germ line stem cell population arises outside of the embryonic gonad and proceeds on a circuitous migration to the germinal epithelium. Specifically, in the avian embryo, the process of germ line stem cell migration proceeds through a series of active and passive migratory phases. The germline stem cells or primordial germ cells (PGCs) located in the epiblast of the unincubated embryo translocate to the hypoblast between stages X-XIII. Subsequently, during gastrulation the PGCs are passively carried by the hypoblast to the germinal crescent at about stage 4. As blood islands develop and the embryo becomes vascularized, the PGCs are passively carried through the blood stream between stages 13-16. Subsequently, the PGCs leave the blood vessels and actively migrate to the gonad. Recently, it has becomes possible to culture germline stem cells using PGC populations from the embryonic blood. The practical application of PGC technology is that it is possible to perform genetic modifications of PGCs in vitro, and transfer the cells to recipient embryos to generate germ-line chimeras. This study evaluates migration of cultured PGCS. Materials and Methods: Three lines of primordial germ cell cultures were established, and continually cultured for over 18 months. To examine the ability of the cultured germ cells to migrate PGCs were loaded with a vital fluorescent dye (PKH-26) and injected into the stage X embryos. At stage 28-30, the gonads were removed and examined for the presence of fluorescent PGCs. Furthermore,injected embryos were cultured through hatching, and grown to sexual maturity. Subsequently, the birds were evaluated from the presence of germline chimerism; and frequency of germline chimerism Results: Variability in the ability of cultured PGCs to migrate to the gonads was discovered. The range of germline chimerism was between 12.5 and 92% for the three cell lines; and the frequency of germline chimeras for any one cell line was between 1.5 and 17%. Conclusion: The results of this study confirm that cultured avian PGCs can retain their ability to migrate to the gonad and produce germline chimeras after long-term culture.