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

ABSTRACT

OBJECTIVE@#To construct the retroviral vector pLEGFP-N1-telomerase reverse transcriptase (TERT) and to investigate the expression of TERT in neonatal mouse hypodermal cells.@*METHODS@#The polymerase chain reaction (PCR)-amplified TERT 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. The TERT 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 expressing EGFP was established. Western blot and immunohistochemical assay showed that the expression level of TERT was significantly elevated in the neonatal mouse hypodermal cells.@*CONCLUSIONS@#A high titer of retrovirus pLEGFP-N1-TERT mediates high-level expression of the exogenous TERT gene in the neonatal mouse hypodermal cells. This protocol has potential applications for skin tissue engineering and cell transplantation therapy.