Development of reconstituted embryos derived from somatic cell nuclei in the rabbit.

Abstract

Production of cloned laboratory animals is helpful in the establishment of medical models. In this study, we examined to produce reconstituted embryos derived from somatic cell nuclei, and to establish embryonic stem (ES) cell lines from the embryo in rabbits. Metaphase II (M-II) oocytes from superovulated rabbit were used as nuclear recipients. Nuclear donor cells were fibroblasts collected from a Dutch Beleted rabbit. The M-II chromosome and the 1st polar body were aspirated, and a fibroblast was inserted into the perivitelline space of the enucleated oocyte. The pairs were electrofused for cell membrane fusion using a cell fusion apparatus, and reconstituted embryos were produced. The embryos were activated and cultured in modified HTF medium and DMEM. The embryos developed to the blastocyst stage were removed their zona pellucida, and they were cultured on the feeder cell layer. As a result of having observed development of reconstituted embryos, 21.2% of the embryos were developed to the blastocyst stage. In the embryos cultured on the feeder cells, the adhesion on feeder cells was observed. We obtained inner cell mass (ICM) colony derived from reconstituted embryos. At present, we are investigating to establish the ES cell lines derived from the embryos reconstituted by nuclear transfer.