Somatic cell nuclear transfer (SCNT) is an efficient technique which has been successfully applied to developmental biology, and resulted in the production of offspring from various species. It offers many opportunities in basic and medical research as well as endangered species preservation. On the other hand, embryonic stem (ES) cells are useful research tools for genetic engineering and developing disease models. In previous study, we established bovine IVF embryo derived ES cell line which can be grow indefinitely as undifferentiated cell state. In this study, we compared the effect of two different age cells (bovine ES cell; JNU-ibES-05 or adult ear fibroblast cell) on in vitro developmental potential of bovine SCNT embryo. To produce SCNT embryos, the ES cells or somatic cells were dissociated and transferred into enucleated MⅡ oocytes, and cleaved reconstructed embryos were cultured in CR1aa medium containing 10% FBS, 1 ug/ml epidermal growth factor (EGF) and 1 ug/ml insulin growth factor (IGF) for 8 days. In the result, blastocyst development rate was similar between ES cell treatment group and somatic cell treatment group, 27.7% (10/36) and 28.9% (11/ 38), respectively. However, there was particular difference in development speed from day 5 post SCNT, blastocyst expanding was 1 day faster in ES cell group than in somatic cell group. This difference was analyzed by semi-quantitative RT-PCR using pluripotency, growth and cell cycle gene markers. These results demonstrated that SCNT embryo using ES cell as a donor cell has better growth potential than somatic cell, and it will be a useful tool for a transgenic animal production.

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate parthenogenetic embryonic stem (ES) cells. Differentiation capacity of the parthenogentic ES cells was rather lower than that of fertilized embryos derived ES cells, which might be the result of the absence of male genome. However, parthenogenetic ES cells might be useful research tool for genetic engineering and generating SCNT embryo derived ES cells. In our previous study, we reported that establishment of several bovine ES cell lines from in vitro fertilized (IVF) embryos named JNU-ibES. Based on this data, the objective of this study is to generate parthenogenetic ES cells and to examine their stem cell characteristics. Total 107 parthenogenetic embryos produced at day 8 or 9 were classified into their developmental stages (full expanded x 40, hatched x 67). For producing ES cells, ICM and trophetoderm-rich clumps were mechanically dissociated and were cultured on mitomycin- C treated mouse embryonic fibroblast feeder cell drop and covered with mineral oil in DMEM medium containing 20% FBS, 5 ng/ml basic FGF, 1% nonessential amino acids, and 0.55 mM b-mercaptoethanol. We obtained 20 primary parthenogenetic bovine ES (pbES)-like cell colonies. And pbES colony formation was higher in hatched blastocyst (25.4%, 16/67) than expanded blastocysts (10%, 4/40). Among those colonies, 5 pbES cell lines were successfully established and they were named as a series of JNU-pbES. These pbES cells were positively expresssed pluripotency markers such as Oct4, Nanog, TRA-1-81, SSEA-1 and alkaline phosphatase. This result demonstrated that the establishment efficiency and characteristics of pbES cell line was very similar to those of ibES cell line.