Expression of highly controllable genes in insect cells using a modified tetracycline-regulated gene expression system

A modified tetracycline-responsive expression system (TRES) for use in inse ct cells was developed. The TRES contains two components: one encodes a tet racycline-controllable transactivator (tTA) and the other contains a tet op erator DNA sequence to drive the luciferase gene. Our results show that the human cytomegalovirus (CMV) promoter, an essential part for strong tTA exp ression in mammalian system, was not functional in insect cells. Thus furth er modifications were required. Functional tTA was efficiently expressed hi Sf9, Sf21, and TN368 cells by the p10 promoter of Autographa californica m ultiple nuclear polyhedrosis virus (AcMNPV) in plasmid form with virus co-i nfection. An increase of up to 258-fold of luciferase activity was detected in these cells when both components in modified TRES were co-transfected. In order to further simplify the experiment, tTA, which is driven by the p1 0 promoter, was inserted into AcMNPV. Luciferase activity was also strongly stimulated by the infection of this tTA expression-recombinant virus with the transfection of a plasmid containing the second TRES component expressi ng luciferase. The luciferase expressions in these systems, either in plasm ids or the tTA gene in virus and luciferase in plasmid, were significantly suppressed by tetracycline. The time course kinetics of tetracycline action to the TRES were further studied. Within a time span of 50 h, the lucifera se activities could be fully suppressed or activated, respectively, corresp onding to the addition or removal of tetracycline. These experiments have e stablished a well-regulated gene expression system for further broad applic ations of molecular biological studies in insect cells. (C) 2000 Published by Elsevier Science B.V. All rights reserved.