minocycline and 4-epianhydrotetracycline

The effects of five tetracycline analogues, anhydrotetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline, on Top10 promoter activity in NT1 tobacco tissue culture cells have been analysed. The concentration that repressed Top10 promoter activity, the level of transgene repression and the kinetics of transgene de-repression were determined for each analogue, and could not be predicted from in vitro binding affinity to the tetracycline repressor or from comparison with animal cells. Doxycycline had the most potent effect on the Top10 promoter and completely inhibited transgene expression at 4 nmol l(-1). Tetracycline was the most versatile of the analogues tested; tetracycline inhibited the Top10 promoter at 10 nmol l(-1) and was easily washed out to restore Top10-driven expression in 12-24 h. A study was also made of the suitability for plant research of a novel tetracycline analogue, GR33076X. In animal cells, GR33076X de-repressed Top10 promoter activity in the presence of inhibitory concentrations of anhydrotetracycline. In NT1, it is shown that GR 33076X can antagonize repression of the Top10 promoter in the presence of tetracycline, but not of anhydrotetracycline or of doxycycline. Different tetracycline analogues can therefore be used to regulate the Top10 promoter in plant cells and this property may be exploited in planning an optimum course of transgene regulation.

Topics: Agrobacterium tumefaciens; Cell Line; Doxycycline; Gene Expression Regulation, Plant; Glucuronidase; Minocycline; Nicotiana; Oxytetracycline; Plants, Genetically Modified; Promoter Regions, Genetic; Recombinant Fusion Proteins; Repressor Proteins; Tetracyclines; Transfection

The tetracycline-regulatable system (TRS) has become a widely adopted tool for modification of gene expression and analysis of gene function in mammalian cells, plants and transgenic animals. We have studied the potential application of the TRS in gene therapy, using a single vector containing both the tetracycline-controlled transactivator (tTA) and the tTA-responsive promoter (tRP) transcribing mouse GM-CSF. Stable 293 cells established using this vector were used to study the kinetics of the TRS in response to various tetracycline analogues. Dose-response studies show that doxycycline is the most potent-analogue in abolishing tTA activity. Kinetic studies indicate that, at 1,000 ng/ml, all the analogues have similar efficiencies in down-regulating the system in given time. In contrast, following the removal of the analogues, there is a temporal, dose-dependent delay in resumption of the tRP activity. The time taken for resumption of near-optimal tRP activity is approximately 48 h for tetracycline, 144 h for anhydrotetracycline, 192 h for minocycline and 216 h for doxycycline when cells were pretreated with 1000 ng/ml of these antibiotics. This property of the analogues can be employed in planning a desired course of transgene regulation.

Topics: Animals; Anti-Bacterial Agents; Cell Line; Dose-Response Relationship, Drug; Doxycycline; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor; Mice; Minocycline; Promoter Regions, Genetic; Tetracycline; Tetracyclines; Time Factors; Transgenes