Chicken Primordial Germ Cell Collection from 5.5 Day Old Embryo Gonad Protocol

- 2006 -

Dissect gonads from 5.5 day old chicken embryos and place them in a 1.5ml siliconized tube containing 500µl of 10% P-FBS DMEM.  After pooling gonads from approximately 10 embryos, centrifuge at 600 x g for 5 minutes.  A 200µl 0.25% trypsin/EDTA (Gibco, Grand Island, NY) solution is then added to the gonad suspension and incubated for 20 minutes at 37ºC.

Upon removal from the incubator, the gonad suspension is teased until only the homogenate remains, which is subsequently removed.  The trypsin is deactivated by the addition of 200µl 20% P-FBS DMEM.  The cell suspension is then passed through a 30 micron filter that has been pre-wet with 50µl of 20% P-FBS DMEM and collected in a 1.5ml siliconized tube.  Rinse the trypsin tube twice with 200µl of 20% P-FBS DMEM and pass through the filter.  Next two washes of 200µl 0% FBS DMEM are passed through the filter.  It may be necessary to remove the filter after the final wash and aspirate the final portion of the suspension that has passed through the filter and add to the collection tube.  Centrifuge the cell suspension for 5 minutes at 600 x g, remove the supernatant, and add 500µl of 20%ES-FBS 199.  The final cell concentration from 10 embryos should be approximately 2 x 10⁶ cells/ml.

Recipes:
0% FBS DMEM
1% Penicillin/Streptomycin
99% Dulbecco’s Modified Eagle’s Medium

10% P-FBS DMEM
10% Premium Fetal Bovine Serum
1% Penicillin/Streptomycin
89% Dulbecco’s Modified Eagle’s Medium

20% P-FBS DMEM
20% Premium Fetal Bovine Serum
1% Penicillin/Streptomycin
79% Dulbecco’s Modified Eagle’s Medium

20%ES-FBS 199
20% Embryonic Stem Cell-Qualified Fetal Bovine Serum
1% Penicillin/Streptomycin
79% Media 199

References:
Moore, D.T., P.H. Purdy, and H.D. Blackburn. 2006. A Method for Preserving Chicken Primordial Germ Cells.  Poult. Sci. 85(10):1784-1790.

Mozdziak, P.E., J. Angerman-Stewart, B. Rushton, S.L. Pardue, and J.N. Petitte. 2005.  Isolation of chicken primordial germ cells using fluorescence-activated cell sorting.  Poult. Sci.  84:594-600.

Obtain information from Harvey Blackburn at: hblackbu@lamar.colostate.edu