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.
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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.
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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
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DOI: https://doi.org/10.1007/s10529-016-2103-6