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

Abstract

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 x 10(8) 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 x 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.

Leukemia Inhibitory Factor and Fibroblast Growth Factor 2 Critically and Mutually Sustain Pluripotency of Rabbit Embryonic Stem Cells

“…The flushing medium was M199 (31100–027; Gibco, Grand Island, NY, USA) supplemented with 3.36 g/L NaHCO 3 (S5761; Sigma), 10% fetal calf serum (26140–079; Gibco) or 20% knockout serum replacement (KSR; Gibco), 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (H3375), and 1% antimycotics (15240; Gibco). Blastocyst or isolated inner cell mass (ICM) plating, recipes for culture media, outgrowth selection, or passage and culturing of ESCs were all based on previous protocols (12,24,25).…”

“…The ESC lines for marker detection were grown on sixwell dishes and rinsed with Dulbecco's phosphate-buffered saline (DPBS) prior to fixation in 4% paraformaldehyde (P-6148; Sigma-Aldrich) solution. At 15 min after fixation at room temperature (RT), ESCs were rinsed with DPBS (three times) prior to AP staining, as previously described (12,24,25). For specific protein marker expressions, ESC colonies were rinsed with DPBS and then fixed in 4% paraformaldehyde 2–4 days after culture.…”

“…The earliest isolation of fertilized embryo-derived rabbit ESC (rESC) lines was reported by Graves and Moreadith (19), followed by Schoonjans et al (49). Recently, many more rESC lines, including those derived from parthenotes (24), have been established (9,12,16,22,25,59).…”

“…The mitotically inactivated mouse embryonic fibroblast (MEF) (9,12,15,16,22,25,54,59) and Sandos inbred mouse embryo-derived thioquanine and ouabain-resistant cells are two types of feeder cells most commonly used in establishing and maintaining ESC lines derived from several species, including mice (50), cattle (54), and humans (42,43). In hESCs, paracrine factors, such as fibroblast growth factor 2 (FGF2), wingless-related mouse mammary tumor virus integration site (Wnt) and/or Activin/Nodal, have been implicated in maintaining cell self-renewal and stemness (1,13,27,54–57,67).…”

Embryonic stem-like cells derived from in vitro produced bovine blastocysts

“…Although very efficient to derivate the human embryonic stem cells (Heins et al, 2004;Kim et al, 2005), the method is dependent on the availability of the species-specific antibody/ antiserum. For example, only recently the antiserum against rabbit embryos was successfully used to isolate the ICM and the putative ES cells (Chiang et al, 2007). However, there is no commercially available antibody/antiserum against bovine embryos.…”