Development of a stable vero cell line constitutively overexpressing hTERT.

Objective: To develop a stable Vero cell line constitutively overexpressing human telomerase reverse transcriptase (hTERT).Methods: The hTERT cDNA was obtained from a recombined plasmid pCI-neo-hTERT,which contained the full length of DNA coding for hTERT by enzyme digestion and introduced into vector phEF/chMAR-hyg,and the constructed recombinant plasmid phEF/chMAR-hyg-hTERT was transfected into Vero cells by liposome transfection.Then,positive clones were screened with hygromycin,and the hTERT gene transcription level of the positive clones was determined by RT-PCR assay and the expression of hTERT gene in high hTERT gene transcription clone was confirmed by Western blotting analysis.The growth and viability of the Vero cells overexpressing hTERT in batch culture in tissue culture plate were examined by Cedex A20 cell counter.Results: A positive clone designated Vero-T1,efficient transcription and expression of hTERT gene was obtained,and the growth of Vero-T1 cells in batch culture was superior to the wild-type Vero cells.Conclusion: A stable Vero cell line constitutively overexpressing hTERT was successfully developed,which laid a foundation for improving both the growth of Vero cells and the vaccine production process by constitutive overexpression of hTERT gene in Vero cells.