Cellular reprogramming of adult goat fibroblast: Toward pluripotency

DOI: 10.18805/ijar.10274    | Article Id: B-3020 | Page : 52-57
Citation :- Cellular reprogramming of adult goat fibroblast: Toward pluripotency .Indian Journal Of Animal Research.2017.(51):52-57

Dharmendra Kumar, Rakesh Ranjan, Ajit P. Singh and Bikash C Sarkhel*

sarkhelbc@yahoo.co.in
Address :

Animal Biotechnology Centre, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, India.

Submitted Date : 20-05-2015
Accepted Date : 17-10-2015

Abstract

Cellular reprogramming erases the epigenetic constraints of somatic cells genome and thus considered as key factor for success of somatic cell nuclear transfer technology. To achieve the reprogramming, different strategies are used which are mostly based on arresting the cell cycle at G0 or G1 stage. The present study was based on molecular investigation of reprogrammed cells for expression of pluripotent genes that are crucial for development of cloned embryos. The fibroblast cell lines were treated by four methods to induce cellular reprogramming viz., serum starvation, Roscovitin, aphidicolin and overconfluent. These treated cell lines were used for quantification of pluripotent gene transcripts by using real time PCR machine. The results showed that the relative expression of different pluripotent genes as Oct-4 and Nanog along with DNA methyl transferase gene (Dnmt-1) was observed in four treated cells. In case of normal cells, only Dnmt-1 gene was expressed, but pluripotent genes were not expressed at detection level. The expression of pluripotent genes in the donor cells prior to nuclear transfer have significant impact on cloning as because it facilitates the expression of that gene in the resulting embryo after nuclear transfer. The finding of this study may be extended for stem cell generation as it showed that pluripotent genes could be induced in the somatic cells without any transgenic incorporation.   

Keywords

Cell cycle Cloning Pluripotent genes Serum starvation Somatic cell.

References

  1. Amarnath, D., Li, X., Kato, Y. and Tsunoda, Y. (2007). Gene expression in individual bovine somatic cell cloned embryos at the 8-cell and blastocyst stages of preimplantation development. Journal of Reproduction and Development. 53:1247-1263. 
  2. Arnold, D.R., Bordigno, V., Lefebvre, R., Murphy, B.D. and Smith, L.C. (2006). Somatic cell nuclear transfer alters peri-    implantation trophoblast differentiation in bovine embryos. Reproduction. 132:279-290.
  3. Beaujean, N., Taylor, J., Gardner, J., Wilmut, I., Meehan, R. and Young, L. (2004). Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer. Biology of Reproduction. 71:185-193.
  4. Campbell, K.H.S., McWhir, J., Ritchie, W.A. and Wilmut, I. (1996). Nuclear equivalence, nuclear transfer, and the cell cycle. Nature. 380:64-66.
  5. Daniels, R., Hall, V.J. and Trounson, A.O. (2000). Analysis of gene transcription in bovine nuclear transfer embryos reconstructed with granulosa cell nuclei. Biology of Reproduction. 63:1034–1040.
  6. Gibbons, J., Arat, S., Rzucidlo, J., Miyoshi, K., Waltenburg, R., Respess, D., Venable, A., Stice, S. (2002). Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells. Biology of Reproduction. 66:895–900.
  7. Hinrichs K., Choi, Y.H., Varner, D.D. and Hartman, D.L. (2007). Production of cloned horse foals using roscovitine-    treated donor cells and activation with sperm extract and/or ionomycin. Reproduction. 134:319–325.
  8. Iyer, V.R., Eisen, M.B., Ross, D.T., Schuler, G., Moore, T.M., Lee, J.C.F., Trent, J.M., Staudt, L.M., Hudson, J., Boguski, M.S., Lashkari, D., Shalon, D., Botstein, D. and Brown, P.O. (1999). The transcriptional program in the response of human fibroblasts to serum. Science. 283:83–87.
  9. Koo, O.J., Hossein, M.S., Hong, S.G., Martinez-Conejero, J.A. and Lee, B.C. (2009). Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer. Zygote. 17:37–43.
  10. Kues, W. A., Anger, M., Carnwath, J.W., Paul, D., Motlik, J. and Niemann, H. (2000). Cell cycle synchronization of porcine fetal fibroblasts: Effects of serum deprivation and reversible cell cycle inhibitors. Biology of Reproduction. 62:412-419.
  11. Lee, J. H. and Campbell, K.H.S. (2006). Effects of enucleation and caffeine on maturation-promoting factor (MPF) and mitogen-activated protein kinase(MAPK) activities in ovine oocytes used as recipient cytoplasts for nuclear transfer. Biology of Reproduction. 74:691-698.
  12. Li, E. (2002). Chromatin modification and epigenetic reprogramming in mammalian development. Nature Reviews Genetics. 3:662–673.
  13. Mayer, W., Niveleau, A., Walter, J., Fundele, R. and Haaf, T. (2000). Demethylation of the zygotic paternal genome. Nature. 403:501–502.
  14. Page, R.L., Ambady, S., Holmes, W.F., Vilner, L., Kole, D., Kashpur, O., Huntress, V., Vojtic, I., Whitton, H. and Dominko, T. (2009). Induction of stem cell gene expression in adult human fibroblasts without transgenes. Cloning and stem cells. 11: 417-26.
  15. Park H.J., Koo, O.J., Kwon, D.K., Kang, J.T., Jang, G. and Lee, B.C. (2009). Effect of roscovitine-treated donor cells on development of porcine cloned embryos. Reprod Dom Anim. doi 10.1111/j.1439-0531.2009.01499.
  16. Rideout III, W. M., Eggan, K. and Jaenisch, R. (2001). Nuclear cloning and epigenetic reprogramming of the genome. Science. 293:1093-1098.
  17. Rodriguez-Alvarez L. and Castro, F.O. (2010). Effect of nucleus transfer on gene expression in bovine embryos during early development. Acta Scientiae Veterinariae. 38:509-519.
  18. Sambrook, J. and Russell, D.W. (2001). Molecular cloning: A laboratory manual. Publishing Cold Spring Harbor Laboratory Press, Newyork. pp. 5.2-5.32.
  19. Shi, D., F. Lu., Wei, Y., Cui, K., Yang, S., Wei, J. and Liu, Q. (2007). Buffalos cloned by nuclear transfer of somatic cells. Biology of reproduction. 77:284-291.
  20. Takahashi K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126:663-676.
  21. Tani, T., Kato, Y. and Tsunoda, Y. (2001). Direct exposure of chromosomes to non activated ovum cytoplasm is effective for bovine somatic cell nucleus reprogramming. Biology of Reproduction. 64:324-330.
  22. Tsai C.C. and Hung, S.C. (2012). Functional roles of pluripotency transcription factors in mesenchymal stem cells. Cell Cycle. 11:3711–3712.
  23. Wang F., Kou, Z., Zhang, Y. and Gao, S. (2007). Dynamic reprogramming of histone acetylation and methylation in the ûrst cell cycle of cloned mouse embryos. Biology of Reproduction. 77:1007–1016. 
  24. Wells, D.N., Labile, G., Tucker, F.C., Miller, A.L., Oliver, J.E., Xiang, T., Forsyth, J.T., Berg, M.C., Cockrem, K., L’Huillier, P.J., Tervit, H.R. and Oback, B. (2003). Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle. Theriogenology. 54:45-59.
  25. Wilmut, I., Schnieke, A.E., Mcwhir, J., Kind, A.J. and Campbell, K.H.S. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature. 385: 810-813.
  26. Xue, F., Tian, X.C., Du, F., Kubota, C., Taneja, M. and Dinnyes, A. (2002). Aberrent patterns of X chromosome inactivation in bovine clones. Nature Genetics. 31:216-220.
     

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