Rabbit cloning by NT with somatic cells is so far a rather inefficient process. However, this technology is urgently required to generate rabbits with a humanized immune system as a source of human polyclonal antibodies. Embryonic stem cells (ESCs) have a number of advantages over somatic cells as tools for cell-mediated transgenesis including long periods of proliferation in vitro, higher frequency of homologous recombination between exogenous and chromosomal DNA, and less requirements for reprogramming (Rideout et al. 2000 Nat. Genet. 24, 109–110). To improve rabbit cloning we have derived and characterized 19 putative rabbit ESC lines and tested cells from 6 lines as donors for NT. First, we assessed in vitro development of NT embryos. Blastocyst rates varied in the range of 6–68% depending on the particular cell line and passage number, but the quality of the resultant embryos was worse compared to NT embryos derived from adult fibroblasts [hatched blastocysts: 13/214 (6%) v. 36/86 (42%), respectively]. Transfer of NT embryos derived from the ESC line showing the highest development to blastocysts into recipients resulted only in implantations (70%, 7/10) but not in offspring. Assuming that poor quality of NT embryos derived from ESCs could be due to the incompatibility between cell cycles of donor and recipient cells we used serum starvation to make ESCs more suitable for nuclear transfer. Serum starvation of one of the ESC lines (0.5% FCS for 3 days) greatly improved the quality of cloned embryos compared to those derived from non-starved cells of the same ESC line as indicated by the high proportions of hatched [38/151 (25%) v. 4/153 (3%)] and attached [25/151 (17%) v. 0%] to the surface of a culture dish blastocysts. Moreover, some of these blastocysts grew in vitro for 14–25 days. Our study provides evidence that the quality of NT embryos derived from ESCs can be significantly improved using serum starvation of donor cells suggesting possible effect of this treatment on the cell cycle synchronization. We are currently testing whether serum starvation of ESCs would also improve post-implantation development of rabbit NT embryos.
This work is supported by Roche Diagnostic GmbH.
Effects of in vitro Culture Period of Reconstructed Embryos and Genetic Background of Feeder Cells on Establishment of Embryonic Stem Cells Derived from Somatic Cell Nuclear Transfer Blastocysts in Pigs
