Production of chicken chimeras from injection of frozen-thawed blastodermal cells

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Abstract
To execute a strategy for reconstituting genetic resources from cryopreserved blastodermal cells, experiments were conducted to optimize conditions for producing chimeric chickens from frozen-thawed blastodermal cells. Stage X blastodermal cells were collected from Barred Plymouth Rock embryos and dispersed. Cells were resuspended in 10% dimethyl sulfoxide in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, and distributed into plastic ampules. Cell suspensions 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. Thawing was done by plunging the ampules into warm water (37 C) for 3 min. After centrifugation, the supernatant was replaced with DMEM, and dead or broken cells were removed by density gradient centrifugation. Approximately 500 cells were injected into irradiated Stage X White Leghorn recipient embryos. Following incubation, several somatic chimeras were produced. The frequency of somatic chimerism when fresh (unfrozen) cells, or cells that were frozen and selected by density gradient centrifugation on Percoll or Nycoprep were injected into recipient embryos was 84, 79, and 85%, respectively. The percentage of donor-derived pigmentation in the down of these chimeric chickens was 79, 50, and 58%, respectively. Germline chimerism was determined by mating the chimeras 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%) injected with cells that were frozen and selected by density gradient centrifugation on a Percoll gradient, and 3 of 26 birds (11.5%) injected with cells that were frozen and selected on a Nycoprep gradient showed germline 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 described here for reconstituting genetic resources from cryopreserved blastodermal cells via chimeric intermediates can be performed successfully.