Construction and identification of retroviral vector phTERT-I-GTKlox for pancreatic β-cell reversible immortalization

Objective To construct a reversible immortalization retroviral vector carrying immortalizing gene human telomerase reverse transcriptase(hTERT)and recombination site sequence loxP.Methods The retroviral vector pBGTKlox carrying EGFP-thymidine kinase fusion gene and loxP sequence was digested by restriction enzyme EcoRⅠ and SalⅠ.IRES sequence was amplified by PCR,and then cloned to the retroviral vector pBGTKlox to establish a new vector pI-GTKlox.The gene coding hTERT was cloned to EcoRⅠ site of pI-GTKlox to establish phTERT-I-GTKlox.phTERT-I-GTKlox was identified by bacterial colony PCR,restriction endonuclease digestion,and DNA sequencing.Results In the five colonies resulted from our experiment,two positive colonies were identified by bacterial colony PCR.As expectably,two fragments of 6.5 kb and 3.5 kb were found in the positive colony plasmid digested by EcoRⅠ.And the cloned hTERT sequence was further verified by DNA sequencing.Conclusion The retroviral vector phTERT-I-GTKlox for reversible immortalization is successfully constructed.Thus it provides an ideal vector in the reversible immortalization of β-cells.