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The proliferative lifespan of adult sheep fibroblasts can be conditionally extended by tetracycline-inducible expression of human telomerase reverse transcriptase

  • Na Zhang
  • Shuo Zhang
  • Hong Guan
  • Jian HouEmail author
Article
  • 24 Downloads

Abstract

Forced expression of human telomerase reverse transcriptase (hTERT) has been used to immortalize mammalian cells. Here, we report conditional extension of proliferative lifespan of adult sheep somatic cells by introducing tetracycline-inducible (Tet-on) expression of hTERT. After transfecting adult sheep fibroblasts with the vector for Tet-on induced conditional expression of hTERT, we obtained several hTERT-positive clones that exhibited extended-lifespan under the induction of tetracycline analogue, doxycycline. Further assays for a representative cell clone A3h38 indicated that the cells had a much stronger proliferative ability than control primary cells, as assessed by population doubling levels and single-cell cloning efficiency. A3h38 cells could maintain vigorous growth in culture for more than 150 days but they became senescent when hTERT expression was abrogated by withdrawal of doxycycline. Although having undergone long-term culture, the nuclei of A3h38 cells could support higher preimplantation development of somatic cell nuclear transfer embryos than control primary cells. These results demonstrated that conditional expression of hTERT could reversibly extend the lifespan of adult sheep fibroblasts, enhance their proliferation and maintain their ability to be reprogrammed after nuclear transfer. This strategy would also be applicable to many other somatic cell types in more species.

Keywords

Human telomerase reverse transcriptase (hTERT) Somatic cell Somatic cell nuclear transfer (SCNT) Sheep Tet-on 

Notes

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31072030) and China Agriculture Research System (Grant No. CARS-39-04).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB (1992) Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA 89:10114–10118CrossRefGoogle Scholar
  2. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279:349–352CrossRefGoogle Scholar
  3. Cui W, Wylie D, Aslam S, Dinnyes A, King T, Wilmut I, Clark AJ (2003) Telomerase-immortalized sheep fibroblasts can be reprogrammed by nuclear transfer to undergo early development. Biol Reprod 69:15–21.  https://doi.org/10.1095/biolreprod.102.013250 CrossRefPubMedGoogle Scholar
  4. Denning C, Dickinson P, Burl S, Wylie D, Fletcher J, Clark AJ (2001) Gene targeting in primary fetal fibroblasts from sheep and pig. Cloning Stem Cells 3:221–231.  https://doi.org/10.1089/15362300152725945 CrossRefPubMedGoogle Scholar
  5. Eun K, Hwang SU, Jeong YW, Seo S, Lee SY, Hwang WS, Hyun SH, Kim H (2017) SV40 large T antigen disrupts embryogenesis of canine and porcine somatic cell nuclear transfer embryo. Proced Online 19:13.  https://doi.org/10.1186/s12575-017-0061-6 CrossRefGoogle Scholar
  6. Fu L, Zhang J, Yan FX, Guan H, An XR, Hou J (2012) Abnormal histone H3K9 dimethylation but normal dimethyltransferase EHMT2 expression in cloned sheep embryos. Theriogenology 78:1929–1938.  https://doi.org/10.1016/j.theriogenology.2012.07.017 CrossRefPubMedGoogle Scholar
  7. He YL, Wu YH, He XN, Liu FJ, He XY, Zhang Y (2009) An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer. Theriogenology 71:1417–1424.  https://doi.org/10.1016/j.theriogenology.2009.01.012 CrossRefPubMedGoogle Scholar
  8. He S, Li Y, Chen Y, Zhu Y, Zhang X, Xia X, Sun H (2016) Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase. Cytotechnology 68:1435–1445.  https://doi.org/10.1007/s10616-015-9903-8 CrossRefPubMedGoogle Scholar
  9. Hong HX, Zhang YM, Xu H, Su ZY, Sun P (2007) Immortalization of swine umbilical vein endothelial cells with human telomerase reverse transcriptase. Mol Cells 24:358–363PubMedGoogle Scholar
  10. Jiang XR, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M, Beeche M, Bodnar AG, Wahl GM, Tlsty TD, Chiu CP (1999) Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet 21:111–114.  https://doi.org/10.1038/5056 CrossRefPubMedGoogle Scholar
  11. Lan GC, Chang ZL, Luo MJ, Jiang YL, Han D, Wu YG, Han ZB, Ma SF, Tan JH (2006) Production of cloned goats by nuclear transfer of cumulus cells and long-term cultured fetal fibroblast cells into abattoir-derived oocytes. Mol Reprod Dev 73:834–840.  https://doi.org/10.1002/mrd.20443 CrossRefPubMedGoogle Scholar
  12. Liu H, Peng H, Liu F, Ma Q, Zhang W (2016) The expression of beta-galactosidase during long-term cultured goat skin fibroblasts and the effect of donor cell passage on in vitro development of nuclear transfer embryos. Vitro Cell Dev Biol Anim 52:555–561.  https://doi.org/10.1007/s11626-015-9984-x CrossRefGoogle Scholar
  13. Morales CP, Holt SE, Ouellette M, Kaur KJ, Yan Y, Wilson KS, White MA, Wright WE, Shay JW (1999) Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat Genet 21:115–118.  https://doi.org/10.1038/5063 CrossRefPubMedGoogle Scholar
  14. Xiang H, Wang J, Mao YW, Li DW (2000) hTERT can function with rabbit telomerase RNA: regulation of gene expression and attenuation of apoptosis. Biochem Biophys Res Commun 278:503–510.  https://doi.org/10.1006/bbrc.2000.3834 CrossRefPubMedGoogle Scholar
  15. Xie Y, Zhao X, Jia H, Ma B (2013) Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase. Vitro Cell Dev Biol Anim 49:8–14.  https://doi.org/10.1007/s11626-012-9554-4 CrossRefGoogle Scholar
  16. Zhang N, Li J, Zhong X, An X, Hou J (2016a) Reversible immortalization of sheep fetal fibroblast cells by tetracycline-inducible expression of human telomerase reverse transcriptase. Biotechnol Lett 38:1261–1268.  https://doi.org/10.1007/s10529-016-2103-6 CrossRefPubMedGoogle Scholar
  17. Zhang S, Guan X, Lu F, Jiang J, Deng Y, Luo C, Shi D (2016b) Establishment and characterization of buffalo fetal fibroblasts induced with human telomerase reverse transcriptase. Theriogenology 86:1622–1629.  https://doi.org/10.1016/j.theriogenology.2016.05.023 CrossRefPubMedGoogle Scholar
  18. Zhao CF, Hu HY, Meng L, Li QQ, Lin AX (2010) Immortalization of bovine mammary epithelial cells alone by human telomerase reverse transcriptase. Cell Biol Int 34:579–586.  https://doi.org/10.1042/cbi20100006 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Agrobiotechnology and College of Biological ScienceChina Agricultural UniversityBeijingChina

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