원문정보
Dual AID and TDG Genes Increase DNA Demethylation of Pluripotency Genes in Bovine Differentiated Cells
초록
영어
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).
목차
INTRODUCTION
MATERIALS AND METHODS
Preparation of bovine somatic cells and cell culture
RNA isolation from bovine tissues and RT-PCR
TA cloning
Construction of expression vector for inducible gene expression
Transfection, selection of cloned cells and cell culture
Alkaline phosphatase (AP) staining
Expression of pluripotency genes
Quantitative real-time PCR
Immunocytochemistry
Western blot
Bisulfite sequencing
Karyotyping
Statistical analysis
RESULTS
Construction of AID and AID+TDG expression vector
Efficiency of transfection and intercellular GFP protein expression of vector
Morphology and AP staining of AID- and AID+TDG inducedbovine cells
Expression of reprogramming and pluripotent genes in transfected cells
Expression of reprogramming factor and pluripotency marker proteins
Expression of pluripotency-related antigen proteins
DNA demethylation in pluripotency gene promoters of in transfected cells
Karyotype
DISCUSSION
REFERENCES