Expression of Human Interferon Alpha2 Gene in Transgenic Cloned Fetus: Recloning in Cattle.

Interferon (IFN) is a protein that has anti-viral and tumor-suppressed effects. It is widely used for therapy of hepatitis, melanoma, and severe acute respiratory syndrome. The current market grows rapidly but current methods of IFN production that uses bacteria cannot satisfy the quantity and quality. This study's goal is producing the transgenic cows that can secrete human IFN-a2 in the milk. Somatic cell nuclear transfer (SCNT) was performed to provide more stable transgene expression. aS1-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 and neomycin resistant was attached to the CMV promoter to allow observation and selection of the donor cell during the experiment. New-born calf fibroblast cells were transfected with lipofectamine and selected by G418. These cells were used for donor cells of SCNT and injected embryos were accomplished by cell to cell fusion. These embryos were then activated with calcium ionomycin and 6-dimethylaminopurine. After 7 days culture in mSOF, blastocysts were used for Embryo Transfer. We tried 90 embryos to 45 surrogate mothers. 8 recipients became pregnant at 50 days of gestation but 7 recipients aborted within 100 days. One fetus kept alive after 200 days but unfortunately on the day 214, this fetus died. We obtained a primary culture of ear fibroblasts from aborted fetus. These fibroblasts matched the donor cells in the Short Tandem Repeat (STR) analysis and differ in the mitochondrial DNA test as expended. Also we confirmed the successful integration of IFN genes and EGFP in these cells through polymerase chain reaction (PCR). As recloning of transgenic somatic cells have been thought to be successfully reprogrammed, fibroblast derived from a transgenic cloned fetus was used for donor cells. In the results, the recloned group had higher blastocyst production (27.9% vs 21.5%) and higher percentage of 50-day pregnancies (28.6% vs 17.8%). We are continuing transfer embryos and waiting for full-term calf. This work was supported by a grant from Next-Generation BioGreen 21 program (No. PJ008054), Rural Development Administration, Republic of Korea.