Expression of Human Erythropoietin Gene in Bovine Somatic Cell Nuclear Transfer Embryos.

Erythropoietin (EPO) is a glycoprotein hormone that controls erythropoiesis. As current methods of EPO production cannot satisfy the demand, cloned animals to be used as bioreactors can help patients with chronic anemia caused by kidney dysfunction. This study was performed to produce human EPO-transgenic cloned cows. Somatic cell nuclear transfer was performed with a vector that was created to induce production of hEPO in cow's milk. AlphaS1-casein was selected as the promoter to be used in this study through the specific promoter activity test, and enhanced green fluorescent protein(EGFP) gene was attached to the CMV promoter to allow observation of the donor cell during the experiment. Two cell lines(calf and adult fibroblast cells) were transfected with lipofectamine. After G418 selection, transfection efficiency (EGFP expression) was 27.3% and 50.0% for calf and adult cells, respectively. The transfected cells were injected to the enucleated oocytes, and injected embryos were accomplished by cell to cell fusion. These embryos were then activated with calcium ionomycin and 6-dimethylaminopurine. Comparing the efficiency of calf and adult fibroblast cells as nuclear transfer donor cells, there were no significant differences in the fusion rate(73.0±10.0% vs. 71.6±12.1%), but in the cleavage rate(63.1±11.7% vs. 56.3±17.1%) and blastocyst rate(16.4±9.0% vs. 14.6±8.0%), the calf cell showed better effects on the embryos(P < 0.05). GFP was expressed in 92.3±2.8% of the blastocysts formed(Calf: 90.4%±3.2 vs Adult: 95.3±2.2%) and the total cell number of blastocysts except the ones used for embryo transfer and DNA sampling was 102.4±22.3(Calf: 99.8±21.4 vs. Adult: 104.6±23.2). We confirmed the successful integration of hEPO genes and EGFP in bovine embryos through polymerase chain reaction (PCR). As the calf fibroblast cell was more efficient than the adult fibroblast cell in nuclear transfer embryo development, we used the calf fibroblast cell as a donor cell for embryo transfer. A total of 58 blastocysts were transferred to 29 surrogate mothers and 4 recipients were found to be pregnant. After a calf is born, microsatellite analysis for determining transgenicity and functional analysis for measuring hEPO in the milk are scheduled. (poster)