doi: 10.15389/agrobiology.2017.2.261eng

UDC 639.122:591.39:57.086.83

Acknowledgements:
Supported financially by Russian Science Foundation (grant № 16-16-04104)

 

ISOLATION, CULTIVATION AND CHARACTERIZATION OF QUAIL PRIMORDIAL GERM CELLS

N.A. Volkova, V.A. Bagirov, E.K. Tomgorova,
A.N. Vetokh, L.A. Volkova, N.A. Zinovieva

L.K. Ernst All-Russian Research Institute of Animal Husbandry, Federal Agency of Scientific Organizations, 60, pos. Dubrovitsy, Podolsk District, Moscow Province, 142132 Russia, e-mail natavolkova@inbox.ru

The authors declare no conflict of interests

ORCID:

Volkova N.A.orcid.org/0000-0001-7191-3550

Vetokh A.N.orcid.org/0000-0002-2865-5960

Bagirov V.A.orcid.org/0000-0001-8385-2433

Volkova L.A.orcid.org/0000-0002-9407-3686

Tomgorova E.K.orcid.org/0000-0001-5398-8815

Zinovieva N.A.orcid.org/0000-0003-4017-6863

Received December 30, 2016

 

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).

Keywords: primordial germ cells, quail, embryos.

 

Full article (Rus)

Full text (Eng)

 

REFERENCES

  1. Petitte J.N., Karagenc L., Ginsburg M. The origin of the avian germ line and transgenesis in birds. Poultry Sci., 1997, 76: 1084-1092 CrossRef
  2. Ginsburg M. Primordial germ cell development in avians. Poultry Sci., 1997, 76: 91-95 CrossRef
  3. Naito M., Tajima A., Tagami T., Yasuda Y., Kuwana T. Preservation of chick primordial germ cells in liquid nitrogen and subsequent production of viable offspring. Journal of Reproduction and Fertility, 1994, 102: 321-325 CrossRef
  4. Naito M., Harumi T., Kuwana T. Long term in vitro culture of chicken primordial germ cells isolated from embryonic blood and incorporation into germline of recipient embryo. J. Poult. Sci., 2010, 47: 57-64 CrossRef
  5. Nakamura Y., Usui F., Miyahara D., Mori T., Ono T., Takeda K., Nirasawa K., Kagami H., Tagami T. Efficient system for preservation and regeneration of genetic resources in chicken: concurrent storage of primordial germ cells and live animals from early embryos of a rare indigenous fowl (Gifujidori). Reprod. Fert. Develop., 2010, 22: 1237-1246 CrossRef
  6. Kuwana T., Kawashima T., Naito M., Yamashita H., Matsuzaki M., Takano T. Conservation of a threatened indigenous fowl (Kureko dori) using the germline chimeras transplanted from primordial germ cells. J. Poult. Sci., 2006, 43: 60-66 CrossRef
  7. Park T.S., Hong Y.H., Kwon S.C., Lim J.M., Han J.Y. Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos. Mol. Reprod. Dev., 2003, 65: 389-395 CrossRef
  8. Chojnacka-Puchta L., Kasperczyk K., Plucienniczak G., Sawicka D., Bednarczyk M. Primordial germ cells (PGCs) as a tool for creating transgenic chickens. Pol. J. Vet. Sci., 2012, 15(1): 181-188 CrossRef
  9. Hong Y.H., Moon Y.K., Jeong D.K., Han J.Y. Improved transfection efficiency of chicken gonadal primordial germ cells for the production of transgenic poultry. Transgenic Res., 1998, 7(4): 247-252 CrossRef
  10. Han J.Y. Germ cells and transgenesis in chickens. Comparative Immunology, Microbiology and Infectious Diseases, 2009, 32: 61-80 CrossRef
  11. Tyack S.G., Jenkins K.A., O’Neil T.E., Wise T.G., Morris K.R., Bruce M.P., McLeod S., Wade A.J., McKay J., Moore R.J., Schat K.A., Lowenthal J.W., Doran T.J. A new method for producing transgenic birds via direct in vivo transfection of primordial germ cells. Transgenic Res., 2013, 22(6): 1257-1264 CrossRef
  12. D’Costa S., Pardue S.L., Petitte J.N. Comparative development of avian primordial germ cells and production of germ line chimeras. Avian Poult. Biol. Rev., 2001, 12(4): 151-168 CrossRef
  13. Kang S.J., Choi J.W., Kim S.Y., Park K.J., Kim T.M., Lee Y.M., Kim H., Lim J.M., Han J.Y. Reproduction of wild birds via interspecies germ cell transplantation. Biol. Reprod., 2008, 79(5): 931-937 CrossRef
  14. Wernery U., Liu C., Baskar V., Guerineche Z., Khazanehdari K.A., Saleem S., Kinne J., Wernery R., Griffin D.K., Chang I.K. Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species. PLoS ONE, 2010, 5: e15824 CrossRef
  15. Yamamoto Y., Usui F., Nakamura Y., Ito Y., Tagami T., Nirasawa K., Matsubara Y., Ono T., Kagami H. A novel method to isolate primordial germ cells and its use for the generation of germlinechimeras in chicken. Biol. Reprod., 2007, 77(1): 115-119 CrossRef
  16. Mozdziak P.E., Angerman-Stewart J., Rushton B., Pardue S.L., Petitte J.N. Isolation of chicken primordial germ cells using fluorescence-activated cell sorting. Poultry Sci., 2005, 84: 594-600 CrossRef
  17. Oishi I. Improvement of transfection efficiency in cultured chicken primordial germ cells by percoll density gradient centrifugation. Biosci. Biotech. Bioch., 2010, 74(12): 2426-2430 CrossRef
  18. van de Lavoir M.C., Diamond J.H., Leighton P.A., Mather-Love C., Heyer B.S., Bradshaw R., Kerchner A., Hooi L.T., Gessaro T.M., Swanberg S.E., Delany M.E., Etches R.J. Germline transmission of genetically modified primordial germ cells. Nature, 2006, 44: 766-769 CrossRef
  19. Macdonald J., Glover J.D., Taylor L., Sang H.M., McGrew M.J. Characterisation and germline transmission of cultured avian primordial germ cells. PLoS ONE, 2010, 5: e15518 CrossRef
  20. Tomgorova E.K., Volkova N.A., Volkova L.A., FisininV.I., Zinovieva N.A. Isolation and characteristics of primordial germ cells in chicken. Agricultural Biology, 2012, 6: 61-65 CrossRef (in Engl.)
  21. Mikroskopicheskaya tekhnika /Pod redaktsiei D.S. Sarkizova, Yu.P. Perova [Microscopy technique. D.S. Sarkizov, Yu.P. Petrov (eds.)]. Moscow, 1996 (in Russ.)..

back