Skip to main content
SpringerLink
Log in

Telomere Elongation Facilitated by Trichostatin A in Cloned Embryos and Pigs by Somatic Cell Nuclear Transfer

  • Published:
Stem Cell Reviews and Reports Aims and scope Submit manuscript

Abstract

Telomere attrition and genomic instability are associated with organism aging. Concerns still exist regarding telomere length resetting in cloned embryos and ntES cells, and possibilities of premature aging of cloned animals achieved by somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), a histone deacetylase inhibitor, effectively improves the developmental competence of cloned embryos and animals, and recently contributes to successful generation of human ntES cells by SCNT. To test the function of TSA on resetting telomere length, we analyzed telomeres in cloned blastocysts and pigs following treatment of SCNT embryos with TSA. Here, we show that telomeres of cloned pigs generated by standard SCNT methods are not effectively restored, compared with those of donor cells, however TSA significantly increases telomere lengths in cloned pigs. Telomeres elongate in cloned porcine embryos during early cleavage from one-cell to four-cell stages. Notably, TSA facilitates telomere lengthening of cloned embryos mainly at morula-blastocyst stages. Knockdown of pTert by shRNA in donor cells reduces telomerase activity in cloned blastocysts but does not abrogate telomere elongation in the TSA-treated embryos (p > 0.05). However, genes associated with recombination or telomerase-independent mechanism of alternative lengthening of telomeres (ALT) Rad50 and BLM show increased expression in TSA-treated embryos. These data suggest that TSA may promote telomere elongation of cloned porcine embryos by ALT. Together, TSA can elongate telomeres in cloned embryos and piglets, and this could be one of the mechanisms underlying improved development of cloned embryos and animals treated with TSA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Shiels, P. G., Kind, A. J., Campbell, K. H., et al. (1999). Analysis of telomere lengths in cloned sheep. Nature, 399, 316–317.

    Article  CAS  PubMed  Google Scholar 

  2. Betts, D., Bordignon, V., Hill, J., et al. (2001). Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle. Proceedings of the National Academy of Sciences of the United States of America, 98, 1077–1082.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Kubota, C., Tian, X. C., & Yang, X. (2004). Serial bull cloning by somatic cell nuclear transfer. Nature Biotechnology, 22, 693–694.

    Article  CAS  PubMed  Google Scholar 

  4. Kurome, M., Hisatomi, H., Matsumoto, S., et al. (2008). Production efficiency and telomere length of the cloned pigs following serial somatic cell nuclear transfer. Journal of Reproduction and Development, 54, 254–258.

    Article  PubMed  Google Scholar 

  5. Jeon, H. Y., Hyun, S. H., Lee, G. S., et al. (2005). The analysis of telomere length and telomerase activity in cloned pigs and cows. Molecular Reproduction and Development, 71, 315–320.

    Article  CAS  PubMed  Google Scholar 

  6. Jiang, L., Carter, D. B., Xu, J., Yang, X., Prather, R. S., & Tian, X. C. (2004). Telomere lengths in cloned transgenic pigs. Biology of Reproduction, 70, 1589–1593.

    Article  CAS  PubMed  Google Scholar 

  7. Lanza, R. P., Cibelli, J. B., Blackwell, C., et al. (2000). Extension of cell life-span and telomere length in animals cloned from senescent somatic cells. Science, 288, 665–669.

    Article  CAS  PubMed  Google Scholar 

  8. Alexander, B., Coppola, G., Perrault, S. D., Peura, T. T., Betts, D. H., & King, W. A. (2007). Telomere length status of somatic cell sheep clones and their offspring. Molecular Reproduction and Development, 74, 1525–1537.

    Article  CAS  PubMed  Google Scholar 

  9. Miyashita, N., Kubo, Y., Yonai, M., et al. (2011). Cloned cows with short telomeres deliver healthy offspring with normal-length telomeres. Journal of Reproduction and Development, 57, 636–642.

    Article  CAS  PubMed  Google Scholar 

  10. Kato, Y., Tani, T., & Tsunoda, Y. (2000). Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows. Journal of Reproduction and Fertility, 120, 231–237.

    CAS  PubMed  Google Scholar 

  11. Jeon, H. Y., Jeong, Y. W., Kim, Y. W., et al. (2012). Senescence is accelerated through donor cell specificity in cloned pigs. International Journal of Molecular Medicine, 30, 383–391.

    CAS  PubMed  Google Scholar 

  12. Dang-Nguyen, T. Q., Haraguchi, S., Akagi, S., et al. (2012). Telomere elongation during morula-to-blastocyst transition in cloned porcine embryos. Cellular Reprogramming, 14, 514–519.

    CAS  PubMed  Google Scholar 

  13. Kishigami, S., Bui, H. T., Wakayama, S., et al. (2007). Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer. Journal of Reproduction and Development, 53, 165–170.

    Article  PubMed  Google Scholar 

  14. Li, J., Svarcova, O., Villemoes, K., et al. (2008). High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos. Theriogenology, 70, 800–808.

    Article  CAS  PubMed  Google Scholar 

  15. Shi, L. H., Ai, J. S., Ouyang, Y. C., et al. (2008). Trichostatin A and nuclear reprogramming of cloned rabbit embryos. Journal of Animal Science, 86, 1106–1113.

    Article  CAS  PubMed  Google Scholar 

  16. Wakayama, S., Kohda, T., Obokata, H., et al. (2013). Successful serial recloning in the mouse over multiple generations. Cell Stem Cell, 12, 293–297.

    Article  CAS  PubMed  Google Scholar 

  17. Tachibana, M., Amato, P., Sparman, M., et al. (2013). Human embryonic stem cells derived by somatic cell nuclear transfer. Cell, 153, 1228–1238.

    Article  CAS  PubMed  Google Scholar 

  18. Hou, M., Wang, X., Popov, N., et al. (2002). The histone deacetylase inhibitor trichostatin A derepresses the telomerase reverse transcriptase (hTERT) gene in human cells. Experimental Cell Research, 274, 25–34.

    Article  CAS  PubMed  Google Scholar 

  19. Schaetzlein, S., Lucas-Hahn, A., Lemme, E., et al. (2004). Telomere length is reset during early mammalian embryogenesis. Proceedings of the National Academy of Sciences of the United States of America, 101, 8034.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Liu, L., Bailey, S. M., Okuka, M., et al. (2007). Telomere lengthening early in development. Nature Cell Biology, 9, 1436–1441.

    Article  CAS  PubMed  Google Scholar 

  21. Ji, G., Liu, K., Chen, C., et al. (2012). Conservation and characterization of unique porcine interstitial telomeric sequences. Science China. Life Sciences, 55, 1029–1037.

    Article  CAS  PubMed  Google Scholar 

  22. Ji, G., Ruan, W., Liu, K., et al. (2013). Telomere reprogramming and maintenance in porcine iPS cells. PLoS One, 8, e74202.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Samstein, B., & Platt, J. L. (2001). Physiologic and immunologic hurdles to xenotransplantation. Journal of the American Society of Nephrology, 12, 182–193.

    CAS  PubMed  Google Scholar 

  24. Vodicka, P., Smetana, K., Jr., Dvorankova, B., et al. (2005). The miniature pig as an animal model in biomedical research. Annals of the New York Academy of Sciences, 1049, 161–171.

    Article  PubMed  Google Scholar 

  25. Lai, L., Park, K. W., Cheong, H. T., et al. (2002). Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells. Molecular Reproduction and Development, 62, 300–306.

    Article  CAS  PubMed  Google Scholar 

  26. Kong, Q. R., Zhu, J., Huang, B., et al. (2011). TSA improve transgenic porcine cloned embryo development and transgene expression. Yi Chuan, 33, 749–756.

    Article  CAS  PubMed  Google Scholar 

  27. Callicott, R. J., & Womack, J. E. (2006). Real-time PCR assay for measurement of mouse telomeres. Comparative Medicine, 56, 17–22.

    CAS  PubMed  Google Scholar 

  28. Cawthon, R. M. (2002). Telomere measurement by quantitative PCR. Nucleic Acids Research, 30, e47.

    Article  PubMed Central  PubMed  Google Scholar 

  29. Jin, H., Wang, J., Wang, F., Ma, J., Mu, Y., & Liu, Z. (2013). Porcine VHL gene cloning and construction of VHL knockdown cloned embryos. Sheng Wu Gong Cheng Xue Bao, 29, 716–725.

    PubMed  Google Scholar 

  30. Ji, G., Liu, K., Okuka, M., Liu, N., & Liu, L. (2012). Association of telomere instability with senescence of porcine cells. BMC Cell Biology, 13, 36.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Muntoni, A., & Reddel, R. R. (2005). The first molecular details of ALT in human tumor cells. Human Molecular Genetics, 14(Spec No. 2), R191–R196.

    Article  CAS  PubMed  Google Scholar 

  32. Bressan, D. A., Baxter, B. K., & Petrini, J. H. (1999). The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae. Molecular and Cellular Biology, 19, 7681–7687.

    CAS  PubMed Central  PubMed  Google Scholar 

  33. Stavropoulos, D. J., Bradshaw, P. S., Li, X., et al. (2002). The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis. Human Molecular Genetics, 11, 3135–3144.

    Article  CAS  PubMed  Google Scholar 

  34. Biessmann, H., & Mason, J. (1997). Telomere maintenance without telomerase. Chromosoma, 106, 63–69.

    Article  CAS  PubMed  Google Scholar 

  35. Huang, J., Wang, F., Okuka, M., et al. (2011). Association of telomere length with authentic pluripotency of ES/iPS cells. Cell Research, 21, 779–792.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Wright, W. E., Piatyszek, M. A., Rainey, W. E., Byrd, W., & Shay, J. W. (1996). Telomerase activity in human germline and embryonic tissues and cells. Developmental Genetics, 18, 173–179.

    Article  CAS  PubMed  Google Scholar 

  37. Blasco, M. A. (2007). The epigenetic regulation of mammalian telomeres. Nature Reviews Genetics, 8, 299–309.

    Article  CAS  PubMed  Google Scholar 

  38. Yamanaka, K., Sugimura, S., Wakai, T., Kawahara, M., & Sato, E. (2009). Acetylation level of histone H3 in early embryonic stages affects subsequent development of miniature pig somatic cell nuclear transfer embryos. Journal of Reproduction and Development, 55, 638–644.

    Article  PubMed  Google Scholar 

  39. Iager, A. E., Ragina, N. P., Ross, P. J., et al. (2008). Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos. Cloning and Stem Cells, 10, 371–379.

    Article  CAS  PubMed  Google Scholar 

  40. Cervera, R. P., Marti-Gutierrez, N., Escorihuela, E., Moreno, R., & Stojkovic, M. (2009). Trichostatin A affects histone acetylation and gene expression in porcine somatic cell nucleus transfer embryos. Theriogenology, 72, 1097–1110.

    Article  CAS  PubMed  Google Scholar 

  41. Maalouf, W. E., Liu, Z., Brochard, V., et al. (2009). Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term. BMC Developmental Biology, 9, 11.

    Article  PubMed Central  PubMed  Google Scholar 

  42. Jarrell, V. L., Day, B. N., & Prather, R. S. (1991). The transition from maternal to zygotic control of development occurs during the 4-cell stage in the domestic pig, Sus scrofa: quantitative and qualitative aspects of protein synthesis. Biology of Reproduction, 44, 62–68.

    Article  CAS  PubMed  Google Scholar 

  43. Tomanek, M., Kopecny, V., & Kanka, J. (1989). Genome reactivation in developing early pig embryos: an ultrastructural and autoradiographic analysis. Anatomy and Embryology (Berlin), 180, 309–316.

    Article  CAS  Google Scholar 

  44. Choi, J. H., Min, N. Y., Park, J., et al. (2010). TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116. Biochemical and Biophysical Research Communications, 391, 449–454.

    Article  CAS  PubMed  Google Scholar 

  45. Sahin, E., Colla, S., Liesa, M., et al. (2011). Telomere dysfunction induces metabolic and mitochondrial compromise. Nature, 470, 359–365.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Park, J. I., Venteicher, A. S., Hong, J. Y., et al. (2009). Telomerase modulates Wnt signalling by association with target gene chromatin. Nature, 460, 66–72.

    Article  CAS  PubMed  Google Scholar 

  47. Takahashi, M. (2012). Oxidative stress and redox regulation on in vitro development of Mammalian embryos. Journal of Reproduction and Development, 58, 1.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant from the National Basic Research Program of China (973 Program, No.932004, 2011CBA01000 and 2009CB941000) and the Fund for Outstanding Young Scholars in Heilongjiang Province (No.JC200905). The authors gratefully acknowledge Bio-X Vision Biological Technology Co., Ltd for Western blot analysis.

Conflict of interest

The authors declare no potential conflicts of interest.

Author information

Authors and Affiliations

  1. Laboratory of Embryo Biotechnology, College of Life Science, Northeast Agricultural University, Harbin, China

    Qingran Kong, Bingteng Xie, Jingyu Li, Juan Wang, Shichao Liu & Zhonghua Liu

  2. Laboratory for Stem Cells and Developmental Biology, College of Life Sciences, Nankai University, Tianjin, China

    Guangzhen Ji, Jian Mao & Lin Liu

  3. State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

    Guangzhen Ji

Authors
  1. Qingran Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guangzhen Ji
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bingteng Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jingyu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jian Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Juan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shichao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhonghua Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Lin Liu or Zhonghua Liu.

Additional information

Qingran Kong and Guangzhen Ji contribute equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1

Telomerase knockdown by RNAi in porcine fibroblasts. A: Significant reduction in pTert mRNA was induced by four pTert shRNAs in TSA-treated porcine fibroblasts. B: sh1 and sh2 co-induced a more efficient reduction of pTert mRNA in TSA-treated porcine fibroblasts. C: Western blot analysis showed pTert protein was effectively knocked down by sh1 and sh2 in TSA-treated porcine fibroblasts. Abbreviations are as follows: C, porcine fibroblasts; T-C, TSA-treated porcine fibroblasts; sh1-sh4, shRNAs; sh1/2, sh1 and sh2; and shC, control shRNA. * indicates p<0.05. (JPEG 12 kb)

High resolution (TIFF 2336 kb)

Supplementary Table 1

(DOCX 17 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kong, Q., Ji, G., Xie, B. et al. Telomere Elongation Facilitated by Trichostatin A in Cloned Embryos and Pigs by Somatic Cell Nuclear Transfer. Stem Cell Rev and Rep 10, 399–407 (2014). https://doi.org/10.1007/s12015-014-9499-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12015-014-9499-y

Keywords

Search

Navigation