Size-dependent isolation of primordial germ cells from avian species

Primordial germ cells (PGCs), the precursors of sperm or ova, could be used to generate transgenic animals and interspecies germ-line chimeras, which would facilitate the recovery of endangered species by making their access and manipulation in vitro easier. During early embryogenesis in avian species, PGCs are transported via the bloodstream to the gonadal anlagen. PGCs of most avian species-particularly wild or endangered birds-are not readily isolated from the embryonic bloodstream because germ-cell markers have not yet been defined for them. Here, we report a rapid, efficient, and convenient method for PGC isolation from various avian species. Blood PGCs were isolated based on the difference in size between PGCs and other blood cells, using a microporous membrane. The efficiency of this size-dependent isolation for the White Leghorn chicken was not significantly different from that of magnetic-activated cell sorting, and the isolated cells expressed chicken PGC-related genes and PGC-specific markers. The utility of the method was then verified in Japanese quail (Coturnix japonica), Mallard duck (Anas platyrhynchos), and Muscovy duck (Cairina moschata). Immunocytochemistry and an in vivo migration assay indicated that this method was able to enrich for true embryonic blood PGCs without specific antibodies, and could be applied to the development of avian interspecies chimeras for restoration of wild or endangered species.