Establishment and identification of tetracycline controlled gene inducible system in immortalized rat astrocyte strains / 中华麻醉学杂志

ABSTRACT

Objective To establish and identify tetracycline controlled gene inducible system in immortalized rat astrocyte strains. Methods The PT67 cells were transfected with pRevTet-On vector. The transfected cells were selected in medium containing G418. RT-PCR assay was used to confirm that Rev Tet-On virus was packed correctly. The viral titer was assayed by infecting NIH3T3 cells. The immortalized astrocyte was infected by RevTet-On virus and individual clones were selected. All individual clones were transiently transfected with pRev TRE-Luc, which carried luciferase gene. The clone with low background and high induction expression was selected and its inducibility was tested. Results The RT-PCR assay showed that RevTet-On virus was packed successfully. The highest viral titer of RevTet-On was 7.4?05 CFU/ml. Rev Tet-On infected immortalized astrocyte and 48 individual clones were isolated. Clone 6 was selected for its highest induction of the luciferase activity in response to doxycycline and the lowest leakiness (activity in the absence of doxycycline) and its inducible fold was 20.6. The expression of luciferase was induced in a dose-dependent manner by doxycycline at the concentrations between 100 and 2 000 ng@ml-1. The expression of luciferase began 1h after doxycycline administration (1000 ng?ml-1) and reached the maximum level 48 h later. Conclusion The tetracycline controlled inducible system is established successfully in immortalized rat astrocyte strains, which is useful for the study of control exogenous gene expression.