Nuclear transfer in rats using an established embryonic cell line and cumulus cells

The present study was performed to establish a rat cell line from blastocysts and to examine the developmental ability of nuclear transfer embryos reconstituted from the established cells and cumulus cells with enucleated mature oocytes. No colonies of embryo-derived cells were observed when morulae were cultured. In contrast, 89% and 40% of the attached embryos formed colonies when blastocysts and hatched blastocysts were cultured, respectively. When the colonies were subcultured in the absence of feeder cells, a cell line with round cell morphology was obtained. This cell morphology was stable up to at least passage 23. The percentage of these embryo-derived cells at G0/G1 was increased when cells were cultured in medium with 0.5% FCS for 48 and 72 h. Ninety-three percent of fresh, non-cultured cumulus cells were at G0/G1. The percentages of reconstructed embryos survival (44-48%), and developed to the 2-cell stage (35%), were significantly higher when the nuclei of cumulus cells were used than when the nuclei of blastocyst-derived cells were used (29% and 3%). The results of the present study indicate that a rat cell line can be established from blastocysts and maintained in vitro for a long period, and that reconstructed embryos obtained by transferring the blastocyst-derived cell nuclei into enucleated oocytes have poorer development potential than those obtained using cumulus cell nuclei. No reconstructed embryos developed beyond the 2-cell stage in vitro.