A single tetracycline-regulated vector devised for controlled insulin gene expression.

Abstract

OBJECTIVE: To construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expression in myotube cell line. METHODS: An expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recombined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentrations of 0, 2, and 10 microg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day. Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits. RESULTS: Immune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation, and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day. CONCLUSION: Human insulin expression can be successfully regulated by doxycycline and the background was very low. This single tet-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.