소 분화세포에서 삽입된 AID, TDG 유전자와 전능성 유전자의 탈메틸화 증가

Abstract

Characteristics of induced pluripotent stem (iPS) cells are consistent with those of embryonic stem (ES) cells. However, exogenous genes integrated by using retrovirus delivery systems cannot be completely removed from the cells. In a recent report, activation-induced cytosine deaminase (AID) and thymine DNA glycosylase (TDG) can induce pluripotency state in mouse differentiated cells through the process of DNA demethylation. Thus, we hypothesized that the two reprogramming factors may convert efficiently bovine differentiated cells into pluripotency state. So, genes of AID and TDG were integrated into pCMV6-AC-IRES-GFP-Puro expression vector, which was transfected into bovine differentiated cells. As results, the colonies derived from AID+TDG-induced bovine cells were formed on day 7 after culture. The number of AP positively colonies in AID+TDG-induced bovine cells was significantly higher than in AID-induced bovine cells (p<0.05). Additionally, expression of pluripotent genes (OCT-3/4, NANOG, SOX2) was slightly increased in AID+TDG-induced bovine cells, as compared to AID-induced bovine cells. Protein expressions of OCT-3/4, NANOG and SOX2 in AID+TDG-induced bovine cells were slightly increased rather than AID-induced bovine cells. Finally, DNA demethylation in the promoter regions of pluripotent markers in AID+TDG-induced bovine cells was increased than that of AID-induced bovine cells. In conclusion, pluripotent stem cells could be efficiently produced from bovine differentiated cells by using non-integrating delivery system with the reprogramming factors (AID and TDG)