Abstract
Objectives
To achieve reversible immortalization of cells, we design a modified tetracycline-inducible expression (Tet-on) system to conditionally regulate the ectopic expression of human telomerase reverse transcriptase (hTERT) in primary cells.
Results
The hTERT gene, hygromycin-resistant gene and all essential elements for achieving tetracycline induction were combined into a single plasmid vector. Sheep fetal fibroblast cells were transfected with the vector and four putative immortalized cell clones were obtained via induction of hTERT expression by doxycycline. These immortalized cells maintained a normal karyotype and showed no transformed phenotype after 250 days continuous culture. When hTERT expression was switched off by withdrawal of doxycycline, the immortalized cells reverted to a normal proliferative state and eventually senesced after limited divisions.
Conclusions
This single-plasmid based Tet-on inducible hTERT expression system can be applied for reversible immortalization of animal cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu C-P, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279:349–352
Clark AJ, Burl S, Denning C, Dickinson P (2000) Gene targeting in livestock: a preview. Transgen Res 9:263–275
Cui W, Aslam S, Fletcher J, Wylie D, Clinton M, Clark AJ (2002) Stabilization of telomere length and karyotypic stability are directly correlated with the level of hTERT gene expression in primary fibroblasts. J Biol Chem 277:38531–38539
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
Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551
Gossen M, Freundlieb S (1995) Transcriptional activation by tetracylines in mammalian cells. Science 268:1766–1769
Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636
Jiang XR, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M, Beeche M, Bodnar AG, Wahl GM, Tlsty TD et al (1999) Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet 21:111–114
Kajiyama H, Titus S, Austin CP, Chiotos K, Matsumoto T, Sakairi T, Kopp JB (2009) Tetracycline-inducible gene expression in conditionally immortalized mouse podocytes. Am J Nephrol 29:153–163
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
Thomas M, Yang L, Hornsby PJ (2000) Formation of functional tissue from transplanted adrenocortical cells expressing telomerase reverse transcriptase. Nat Biotechnol 18:39–42
Wei P, Li L, Qi H, Zhou HX, Deng CY, Li FR (2012) Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells. Biochem Biophys Res Commun 418:330–335
Xiang H, Wang J, Mao YW, Li DC (2000) hTERT can function with rabbit telomerase RNA: regulation of gene expression and attenuation of apoptosis. Biochem Biophys Res Commun 278:503–510
Acknowledgments
This research was supported by the National Natural Science Foundation of China (Grant No. 31072030) and the Earmarked Fund for Modern Agro-industry Technology Research System (Grant No. CARS-40-08).
Supporting information
Supplementary Table 1—Primers used.
Supplementary Fig. 1—The structure of the plasmid pTet-on EGFP.
Supplementary Fig. 2—Proliferative capacity of the hTERT transgenic cells.
Supplementary Fig. 3—The reversal of immortalized cells.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhang, N., Li, J., Zhong, X. et al. Reversible immortalization of sheep fetal fibroblast cells by tetracycline-inducible expression of human telomerase reverse transcriptase. Biotechnol Lett 38, 1261–1268 (2016). https://doi.org/10.1007/s10529-016-2103-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-016-2103-6