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DNA methyltransferase 3a is Correlated with Transgene Expression in Transgenic Quails

  • Jang, Hyun-Jun (WCU Biomodulation Major Department of Agricultural Biotechnology, Seoul National University) ;
  • Kim, Young-Min (WCU Biomodulation Major Department of Agricultural Biotechnology, Seoul National University) ;
  • Rengaraj, Deivendran (WCU Biomodulation Major Department of Agricultural Biotechnology, Seoul National University) ;
  • Shin, Young-Soo (Department of Animal Science, Shingu University) ;
  • Han, Jae-Yong (WCU Biomodulation Major Department of Agricultural Biotechnology, Seoul National University)
  • Received : 2011.06.03
  • Accepted : 2011.06.14
  • Published : 2011.06.30

Abstract

DNA methyltransferases (DNMTs) are closely associated with the epigenetic change and the gene silencing through the regulation of methylation status in animal genome. But, the role of DNMTs in transgene silencing has remained unclear. So, we examined whether the knockdown of DNMT influences the reactivation of transgene expression in the transgenic quails. In this study, we investigated the expression of DNMT3a, and DNMT3b in blastoderm, quail embryonic fibroblasts (QEFs) and limited embryonic tissues such as gonad, kidney, heart and liver of E6 transgenic quails (TQ2) by RT-PCR. We further analyzed the expression of DNMT3a at different stages of whole embryos during early embryonic development by qRT-PCR. DNMT3a expression was detected in all test samples; however, it showed the highest expression in E6 whole embryo. Embryonic fibroblasts collected from TQ2 quails were treated with two DNMT3a-targeted siRNAs (siDNMT3a-51 and siDNMT3a-88) for RNA interference assay, and changes in expression were then analyzed by qRT-PCR. The siDNMT3a-51 and siDNMT3a-88 reduced 53.34% and 64.64% of DNMT3a expression in TQ2 QEFs, respectively. Subsequently the treatment of each siRNA reactivated enhanced green fluorescent protein (EGFP) expression in TQ2 (224% and 114%). Our results might provide a clue for understanding the DNA methylation mechanism responsible for transgenic animal production and stable transgene expression.

References

  1. Ballestar, E. and Esteller, M. 2008. SnapShot: the human DNA methylome in health and disease. Cell. 135(6):1144-1144 e1141. https://doi.org/10.1016/j.cell.2008.11.040
  2. Bernstein, B. E., Meissner, A. and Lander, E. S. 2007. The mammalian epigenome. Cell. 128(4):669-681. https://doi.org/10.1016/j.cell.2007.01.033
  3. Champagne, F. A. 2011. Maternal imprints and the origins of variation. Horm Behav. 60(1):4-11. https://doi.org/10.1016/j.yhbeh.2011.02.016
  4. Chedin, F. 2011. The DNMT3 Family of Mammalian De Novo DNA Methyltransferases. Prog Mol Biol Transl Sci. 101:255-285. https://doi.org/10.1016/B978-0-12-387685-0.00007-X
  5. Chen, T. and Li, E. 2004. Structure and function of eukaryotic DNA methyltransferases. Curr Top Dev Biol. 60:55-89. https://doi.org/10.1016/S0070-2153(04)60003-2
  6. Escors, D. and Breckpot, K. 2010. Lentiviral vectors in gene therapy: their current status and future potential. Arch Immunol Ther Exp (Warsz). 58(2):107-119. https://doi.org/10.1007/s00005-010-0063-4
  7. Fatemi, M., Pao, M. M., Jeong, S., Gal-Yam, E. N., Egger, G., Weisenberger, D. J. and Jones, P. A. 2005. Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level. Nucleic Acids Res. 33(20):e176. https://doi.org/10.1093/nar/gni180
  8. Feinberg, A. P. and Tycko, B. 2004. The history of cancer epigenetics. Nat Rev Cancer. 4(2):143-153. https://doi.org/10.1038/nrc1279
  9. Gorman, C. M., Merlino, G. T., Willingham, M. C., Pastan, I. and Howard, B. H. 1982. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 79(22):6777-6781. https://doi.org/10.1073/pnas.79.22.6777
  10. Hotta, A. and Ellis, J. 2008. Retroviral vector silencing during iPS cell induction: an epigenetic beacon that signals distinct pluripotent states. J Cell Biochem. 105(4):940-948. https://doi.org/10.1002/jcb.21912
  11. Jang, H. J., Choi, J. W., Kim, Y. M., Shin, S. S., Lee, K. and Han, J. Y. 2011. Reactivation of transgene expression by alleviating CpG methylation of the Rous sarcoma virus promoter in transgenic Quail cells. Mol Biotechnol. DOI 10.1007/s12033-011-9393-7 https://doi.org/10.1007/s12033-011-9393-7
  12. Kim, J. N., Park, T. S., Park, S. H., Park, K. J., Kim, T. M., Lee, S. K., Lim, J. M. and Han, J. Y. 2010. Migration and proliferation of intact and genetically modified primordial germ cells and the generation of a transgenic chicken. Biol Reprod. 82(2):257-262. https://doi.org/10.1095/biolreprod.109.079723
  13. Kinney, S. R. and Pradhan, S. 2011. Regulation of expression and activity of DNA (Cytosine-5) methyltransferases in Mammalian cells. Prog Mol Biol Transl Sci. 101:311-333. https://doi.org/10.1016/B978-0-12-387685-0.00009-3
  14. Koo, B. C., Kwon, M. S., Choi, B. R., Lee, H. T., Choi, H. J., Kim, J. H., Kim, N. H., Jeon, I., Chang, W. and Kim, T. 2004. Retrovirus-mediated gene transfer and expression of EGFP in chicken. Mol Reprod Dev. 68(4):429-434. https://doi.org/10.1002/mrd.20102
  15. Kwon, S. C., Choi, J. W., Jang, H. J., Shin, S. S., Lee, S. K., Park, T. S., Choi, I. Y., Lee, G. S., Song, G. and Han, J. Y. 2010. Production of biofunctional recombinant human interleukin 1 receptor antagonist (rhIL1RN) from transgenic quail egg white. Biol Reprod. 82(6):1057-1064. https://doi.org/10.1095/biolreprod.109.081687
  16. Lee, S. I., Kim, J. K., Park, H. J., Jang, H. J., Lee, H. C., Min, T., Song, G. and Han, J. Y. 2010. Molecular cloning and characterization of the germ cell-related nuclear orphan receptor in chickens.Mol Reprod Dev. 77(3):273-284.
  17. Li, E., Bestor, T. H. and Jaenisch, R. 1992. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 69(6):915-926. https://doi.org/10.1016/0092-8674(92)90611-F
  18. Li, E., Bestor, T. H. and Jaenisch, R. 1992. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 69(6):915-926. https://doi.org/10.1016/0092-8674(92)90611-F
  19. Livak, K. J. and Schmittgen, T. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25(4):402-408. https://doi.org/10.1006/meth.2001.1262
  20. Mizuarai, S., Ono, K., Yamaguchi, K., Nishijima, K., Kamihira, M. and Iijima, S. 2001. Production of transgenic quails with high frequency of germ-line transmission using VSV-G pseudotyped retroviral vector. Biochem Biophys Res Commun. 286(3):456-463. https://doi.org/10.1006/bbrc.2001.5422
  21. Okano, M., Bell, D. W., Haber, D. A. and Li, E. 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 99(3): 247-257. https://doi.org/10.1016/S0092-8674(00)81656-6
  22. Overbeek, P. A., Lai, S. P., Van Quill, K. R. and Westphal, H. 1986. Tissue-specific expression in transgenic mice of a fused gene containing RSV terminal sequences. Science. 231(4745): 1574-1577. https://doi.org/10.1126/science.3006249
  23. Park, S. H., Kim, J. N., Park, T. S., Lee, S. D., Kim, T. H., Han, B. K. and Han, J. Y. 2010. CpG methylation modulates tissue-specific expression of a transgene in chickens. Theriogenology. 74(5):805-816 e801. https://doi.org/10.1016/j.theriogenology.2010.04.005
  24. Pearson, R., Kim, Y. K., Hokello, J., Lassen, K., Friedman, J., Tyagi, M. and Karn, J. 2008. Epigenetic silencing of human immunodeficiency virus (HIV) transcription by formation of restrictive chromatin structures at the viral long terminal repeat drives the progressive entry of HIV into latency. J Virol. 82(24):12291-12303. https://doi.org/10.1128/JVI.01383-08
  25. Rengaraj, D., Lee, B. R., Lee, S. I., Seo, H. W. and Han, J. Y. 2011. Expression patterns and miRNA regulation of DNA methyl- transferases in chicken primordial germ cells. PLoS One. 6(5):e19524. https://doi.org/10.1371/journal.pone.0019524
  26. Seo, H. W., Rengaraj, D., Choi, J. W., Ahn, S. E., Song, Y. S., Song, G. and Han, J. Y. 2010. Claudin 10 is a glandular epithelial marker in the chicken model as human epithelial ovarian cancer. Int J Gynecol Cancer. 20(9):1465-1473.
  27. Shin, S. S., Kim, T. M., Kim, S. Y., Kim, T. W., Seo, H. W., Lee, S. K., Kwon, S. C., Lee, G. S., Kim, H., Lim, J. M. and Han, J. Y. 2008. Generation of transgenic quail through germ cell-mediated germline transmission. FASEB J. 22(7):2435-2444. https://doi.org/10.1096/fj.07-101485
  28. Zhang, P. J., Hayat, M., Joyce, C., Gonzalez-Villasenor, L. I., Lin, C. M., Dunham, R. A., Chen, T. T. and Powers, D. A. 1990. Gene transfer, expression and inheritance of pRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (Linnaeus). Mol Reprod Dev. 25(1):3-13. https://doi.org/10.1002/mrd.1080250103