PRODUCTION OF CHICKEN CHIMERAS FROM INJECTION OF FROZEN-THAWED BLASTODERMAL CELLS

To execute a strategy for reconstituting genetic resources from cryopr eserved blastodermal cells, experiments were conducted to optimize con ditions for producing chimeric chickens from frozen-thawed blastoderma l cells. Stage X blastodermal cells were collected from Barred Plymout h Rock embryos and dispersed. Cells were resuspended in 10% dimethyl s ulfoxide in Dulbecco's modified Eagle's medium (DMEM) containing 20% f etal bovine serum, and distributed into plastic ampules. Cell suspensi ons were seeded to induce ice formation at -7 C, cooled from -7 to -35 C at 1 C/min and then ampules were plunged into liquid nitrogen. Thaw ing was done by plunging the ampules into warm water (37 C) for 3 min. After centrifugation, the supernatant was replaced with DMEM, and dea d or broken cells were removed by density gradient centrifugation. App roximately 500 cells were injected into irradiated Stage X White Legho rn recipient embryos. Following incubation, several somatic chimeras w ere produced. The frequency of somatic chimerism when fresh (unfrozen) cells, or cells that were frozen and selected by density gradient cen trifugation on Percoll or Nycoprep were injected into recipient embryo s was 84, 79, and 85%, respectively. The percentage of donor-derived p igmentation in the down of these chimeric chickens was 79, 50, and 58% , respectively. Germline chimerism was determined by mating the chimer as that survived to sexual maturity to Barred Plymouth Rocks. Nine of 16 birds (56.2%) injected with fresh cells, 2 of 26 birds (7.7%) injec ted with cells that were frozen and selected by density gradient centr ifugation on a Percoll gradient, and 3 of 26 birds (11.5%) injected wi th cells that were frozen and selected on a Nycoprep gradient showed g ermline transmission; the percentage of donor-derived progeny in these chimeras were 29.5, 5.2, and 6.8%, respectively. The Barred Plymouth Rock donor stock was ''reconstituted'' by inter se mating of germline male and female chimeras. These data demonstrate that the strategy des cribed here for reconstituting genetic resources from cryopreserved bl astodermal cells via chimeric intermediates can be performed successfu lly.