Earticle

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

ABSTRACT
 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
 

저자

• 진상진 [ Sang-Jin Jin | 경상대학교 농업생명과학대학 ]
• 백상기 [ Sang-Ki Baek | 경상대학교 농업생명과학대학 ]
• 조영수 [ Young-Soo Cho | 경상대학교 농업생명과학대학 ]
• 문송이 [ Song-Yi Moon | 경상대학교 축산생명학과 동물생명과학전공 ]
• 김태숙 [ Tae-Suk Kim | 경상대학교 축산생명학과 동물생명과학전공 ]
• 이준희 [ Joon-Hee Lee | 경상대학교 축산생명학과 동물생명과학전공 ] Corresponding author

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