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Molecular Biotechnology

, Volume 49, Issue 3, pp 222–228 | Cite as

Reactivation of Transgene Expression by Alleviating CpG Methylation of the Rous sarcoma virus Promoter in Transgenic Quail Cells

  • Hyun-Jun Jang
  • Jin Won Choi
  • Young Min Kim
  • Sang Su Shin
  • Kichoon Lee
  • Jae Yong Han
Research

Abstract

In this study, we investigated the relative expression of the Rous sarcoma virus (RSV) promoter-driven expression of enhanced green fluorescent protein (EGFP) in fibroblasts of transgenic quails. We analyzed the direct influence of CpG methylation of the RSV promoter on the transcriptional activity of delivered transgenes. Embryonic fibroblasts collected from homozygous transgenic quail (TQ2) were treated with 50 μM of DNA methyltransferase inhibitor followed by 5-aza-2′-deoxycytidine (5-azadC) for 48 h, and changes in expression were then analyzed by flow cytometry. The results show a significant increase of EGFP expression in TQ2 embryonic fibroblasts (QEFs) (2.64% to 79.84%). Subsequent methylation-specific amplification revealed that 5-azadC significantly reduced the CpG methylation status in the RSV promoters of the QEFs (86.42 to 48.41%); even after 5-azadC was withdrawn, CpG methylation remained decreased in expanded culture (16.28%). Further analysis showed that potential transcription factor binding sites existed in the CpG methylation site of the RSV promoter. These results may provide the basis for understanding the epigenetic mechanism responsible for transgenic animal production and genetic preservation.

Keywords

Transgenic quail RSV promoter Transgene expression CpG methylation 5-aza-2′-deoxycytidine 

Abbreviations

RSV

Rous sarcoma virus

LTR

Long terminal repeat

5-azadC

5-aza-2′-deoxycytidine

QEF

Quail embryonic fibroblast

EGFP

Enhanced green fluorescent protein

Notes

Acknowledgments

This research was supported by the WCU (World Class University) program (R31-10056) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology and the Korea Research Foundation (KRF-2009-220- F00006).

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Hyun-Jun Jang
    • 1
  • Jin Won Choi
    • 1
  • Young Min Kim
    • 1
  • Sang Su Shin
    • 2
  • Kichoon Lee
    • 2
  • Jae Yong Han
    • 1
  1. 1.Department of Agricultural Biotechnology, WCU Biomodulation MajorSeoul National UniversityGwanak-gu SeoulKorea
  2. 2.Department of Animal SciencesThe Ohio State UniversityColumbusUSA

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