Successful Induction of Antisera Against Rabbit Embryos for Isolation of the ICM and Putative Embryonic Stem Cells

In Expt 1, goat antisera against rabbit blastocysts were induced using spleen cell injection and skin-graft for immunosurgical isolation of ICM cells. Goats received rabbit spleen cell suspension (4 × 10⁸ cells/ml) intravenously once a week for three consecutive weeks, plus an additional dose (boost injection) 10 days after the third injection, or a piece of rabbit skin (3 × 3 cm) transplantation. Blood samples were collected starting from the day after the last cell injection for 21 days. Serum was separated, heat inactivated and stored in frozen condition before titre analysis. Results showed that the antisera/antibodies derived by spleen cell injection reached their peak titre 7 days after the last cell injection, compared with 5 days by the skin-grafted group. In Expt 2, morphologically normal blastocysts were collected for isolating ICMs immunosurgically or for direct culture of zona-free whole blastocysts. In both methods, ICM cells started attaching to the feeder layer and outgrowing from the centre portion of the cells on day 3 after the onset of culture. ICM outgrowths increased in size during days 4–5, and most cells differentiated morphologically after day 6. One colony derived from isolated ICM developed into morphologically ES-like cells expressing alkaline phosphatase activity. Our results indicated that both skin-grafting and spleen cell injection were effective inducing antisera against rabbit embryonic cells. More studies are required to optimize the culture system for rabbit ES cells.