Delay in resumption of the activity of tetracycline-regulatable promoter following removal of tetracycline analogues

Abstract

The tetracycline-regulatable system (TRS) has become a that, at 1000 ng/ml, all the analogues have similar widely adopted tool for modification of gene expression efficiencies in down-regulating the system in a given time. and analysis of gene function in mammalian cells, plantsIn contrast, following the removal of the analogues, there and transgenic animals. We have studied the potential is a temporal, dose-dependent delay in resumption of the application of the TRS in gene therapy, using a single vectRP activity. The time taken for resumption of near-optimal tor containing both the tetracycline-controlled transactivtRP activity is approximately 48 h for tetracycline, 144 h for ator (tTA) and the tTA-responsive promoter (tRP) transcribanhydrotetracycline, 192 h for minocycline and 216 h for ing mouse GM-CSF. Stable 293 cells established using doxycycline when cells were pretreated with 1000 ng/ml of this vector were used to study the kinetics of the TRS in these antibiotics. This property of the analogues can be response to various tetracycline analogues. Doseemployed in planning a desired course of transgene reguresponse studies show that doxycycline is the most potentlation. analogue in abolishing tTA activity. Kinetic studies indicate

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

“…Several features, for example, a high induction capacity and rapid responsiveness, make the Tet-On system particularly attractive as an in vivo inducible system. 1,7 The in vivo consequences of long-term exposure to antibiotics-such as Tc and dox-also have to be taken into account. Using the Tet-On approach, the extent of exposure to dox is rather limited when expression of the target gene is required only for a short period of time.…”

Optimization of the Tet-On system for regulated gene expression through viral evolution

“…As longterm exposure to effectors is often undesirable, the Tet-On system is preferred in applications in which gene expression is to be sustained in a switched-off state for long periods, or when rapid gene induction is required. 6,7 Unfortunately, the amino-acid substitutions in rtTA that confer the reverse phenotype also affect its overall performance. As a consequence, rtTA has lost the ability to be activated by Tc and other dox-like compounds, and it requires 100-fold more dox for maximal induction than that is needed for tTA inhibition.…”

Regulatable systems: applications in gene therapy and replicating viruses

“…In addition, certain tetracycline analogs, such as Dox, crosses placental barrier and are also secreted into milk, making this system particularly suitable for transgenic work. The TrRS is also distinguished from the other regulatable systems by its impressive degree of regulation and the ability to alter the kinetics of the response using different tetracycline analogs (16). Moreover, the system can be switched off and then switched on within 72 hours when using oxytetracycline (16).…”

“…The TrRS is also distinguished from the other regulatable systems by its impressive degree of regulation and the ability to alter the kinetics of the response using different tetracycline analogs (16). Moreover, the system can be switched off and then switched on within 72 hours when using oxytetracycline (16). A final, unique advantage lies in the fact that, whereas all other chimeric systems either induce or suppress transgene expression, the tetracycline system can be engineered to work in either direction or in both ways simultaneously (17)(18)(19).…”