title: Reversible immortalization of sheep fetal fibroblast cells by tetracycline-inducible expression of human telomerase reverse transcriptase.
abstract: OBJECTIVE
  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.
authors.affiliation: State Key Laboratory of Agrobiotechnology and College of Biological Science, 58# Center for Life Sciences, China Agricultural University, Yuan-Ming-Yuan West Road, Haidian District, Beijing, 100193, China.
authors.forename: Na
authors.initials: N
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agrobiotechnology and College of Biological Science, 58# Center for Life Sciences, China Agricultural University, Yuan-Ming-Yuan West Road, Haidian District, Beijing, 100193, China.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agrobiotechnology and College of Biological Science, 58# Center for Life Sciences, China Agricultural University, Yuan-Ming-Yuan West Road, Haidian District, Beijing, 100193, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Zhong
authors.affiliation: State Key Laboratory of Agrobiotechnology and College of Biological Science, 58# Center for Life Sciences, China Agricultural University, Yuan-Ming-Yuan West Road, Haidian District, Beijing, 100193, China.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: An
authors.affiliation: State Key Laboratory of Agrobiotechnology and College of Biological Science, 58# Center for Life Sciences, China Agricultural University, Yuan-Ming-Yuan West Road, Haidian District, Beijing, 100193, China. houjian@cau.edu.cn.
authors.forename: Jian
authors.initials: J
authors.lastname: Hou
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology*; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D012756:Sheep; D019098:Telomerase / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology*
chemical_list: C509186:TERT protein, human; D019098:Telomerase; D013752:Tetracycline
keywords: Genetic modification in animals; Human telomerase reverse transcriptase; Immortalization; Sheep; Somatic cells; Telomerase repeat amplification protocol; Tetracycline-inducible expression