Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cel

Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cells for

skin tissue engineering

Ting Tan;Zhi-Qi Hu

【期刊名称】《亚太热带医药杂志（英文版）》 【年(卷),期】2013(000)012

【摘要】Objective:To construct the retroviral vector pLEGFP-N1-telomerase reverse transcriptase(TERT) and to investigate the expression ofTERT in neonatal mouse hypodermal cells.Methods:The polymerase chain reaction(PCR)-amplifiedTERT gene was inserted into plasmid pLEGFP-N1.The positive clone was identified by restriction enzyme digestion and sequencing, then was transfected into packaging cells to produce retrovirus particles.Neonatal mouse hypodermal cells were infected with the virus to generate a stable cell line.TheTERT mRNA expression level, telomerase activity, and enhanced green fluorescent protein(EGFP) expression level were analyzed.Results:Retroviral vector pLEGFP-N1-TERT was constructed successfully, and a stable cell line of neonatal

mouse

hypodermal

cells

expressingEGFP

was

established.Western blot and immunohistochemical assay showed that the expression level ofTERT was significantly elevated in the neonatal mouse hypodermal cells.Conclusions:A high titer of retrovirus pLEGFP-N1-TERT mediates high-level expression of the exogenousTERT gene in