Isolation, cultivation and characterization of quail primordial germ cells

The use of avian primordial germ cells (PGCs) for production of chimeric and transgenic poultry is regarded as an alternative way to traditional methods of selection and transgenesis. This approach involves the introduction of donor primordial germ cells in the dorsal aorta of the recipient embryo during their migration from blood to gonads. In case of recipient embryo gonads colonization by donor PGCs, the further differentiation of donor cells into mature germ cells becomes possible, for both males and females. A key factor for the effectiveness of this manipulation is obtaining of a pure population of embryonic cells. In this regard, the development of effective methods for isolation and maintenance of PGCs in culture is important. Our research was aimed at the improvement of methodical approaches for isolation and cultivation of quail PGCs. This type of cells was isolated and characterized. Five- to six-day embryos were used for obtaining PGCs culture. Isolation of PGCs from quail embryos was performed using two methodological approaches - mechanical dissociation and enzymatic treatment. Trypsin solution at concentration from 0.05 % to 0.25 % was used as proteolytic enzyme for enzymatic treatment. In order to obtain the most pure populations of PGCs, unequal ability to adhesion of different types of cells was taken into account. It was found that enzymatic treatment with 0.05 % trypsin is an effective method for embryos disaggregation preserving significant proportion of viability (94 %) of fetal cells. Separation of different cell types based on their different ability to adhesion allows obtaining PGCs culture maximally purified from other cell types. Moreover, single embryo fibroblasts, remaining in the PGCs cell suspension after separation from the other cell types are used as a feeder layer to which PGCs are attached at the subsequent cultivation. It was shown that own primary embryonic fibroblasts are optimal as a feeder layer for short-term PGCs culturing as compared to the use of STO cells and cultured embryonic fibroblasts. If using the growth medium based on DMEM with high glucose level (4.5 g/l) supplemented with 20 % fetal bovine serum, 2 mM glutamine, 10-6 mM 2-mercaptoethanol, 2 ng/ml LIF (leukemia inhibitory factor), 10 mM essential amino acids (MEM), the antibiotic gentamicin (50 ug/ml) the attachment of PGCs to the feeder layer was observed at day 1 to 2 of cultivation forming colonies at day 3 to 4. The presence of primordial germ cell colonies was confirmed by immunohistochemistry using specific primary antibodies to SSEA-1 (stage-specific embryonic antigen-1).