What are Inducible Expression Vectors?
Inducible Expression Vector help you in gene manipulation, this is done by spatial-temporally controlling gene expression especially in mammalian cell. This system is adopted from bacteria which are tetracycline resistant.
The tetracycline inducible system constitutes of two components, a tetracycline dependent promoter and a tetracycline-controlled regulator.
A tetracycline-dependent promoter is developed by placing a tetracycline response element (TRE) upstream of a minimal promoter. A TRE is a series of 7 repeats of a 19-nucleotide tetracycline operator (tetO). It can be recognized by the tetracycline repressor (tetR) in the endogenous bacterial system, which then inhibits the transcription of downstream gene. In the presence of tetracycline, tetR will bind to tetracycline and not to TRE. The transcription of the gene will not be inhibited by the repressor.
OriGene's Inducible Gene Expression Solution
OriGene's All-in-one Tet-ON system is a new and improved version of the original Tet-ON systems designed to significantly stimulate expression of the downstream gene of interest (GOI). It has a Tet-On 3G transactivator and a tightly regulated TRE promoter (PTIGHT) in one vector.
|PS100125||Turbo-GFP tag, Tet-ON 3G transactivator|
|PS100124||Myc-DDK tag, Tet-ON 3G transactivator|
The Tet-ON 3G transactivator consists of a modified bacterial Tet repressor (TetR) fused to three minimal VP16 activation domains to create a transcriptional activator protein. Our Tet-ON 3G transactivator contains mutations that significantly increase its sensitivity to Doxycycline (Dox), a synthetic analog to tetracycline.
The increased sensitivity is particularly advantageous for in vivo studies in tissues where high Dox concentrations are difficult to attain (e.g., brain). The tightly regulated PTIGHT promoter consists of the conventional TRE sequence fused upstream of the minimal CMV promoter which provides remarkably low basal activity and high maximal expression after induction. The significantly reduced background expression provides an improved dynamic expression range compared to traditional Tet promoter.
Application of Tet-On Vectors
- Tight and Timely regulation of target gene expression
- Investigate the role of your gene of interest in progression of diseases, cell cycle, or tissue growth.
Significant induction of GFP expression was observed in HEK 293 cells transfected with Tet-On vector at Dox dosage of 1ug/ml or higher, 72 hours post treatment, as shown in the microscopic images. Concomitantly, we performed a quantitative measurement of the induction of the GFP expression by Dox on the same cells using a fluorescence plate reader at 0, 24, 48, and 72 hours post Dox treatment.
Raw fluorescence intensity readout (expressed in arbitrary units) from the plate reader plotted against varying concentrations of Dox within their respective days clearly demonstrates the most effective dosage to be at 1ug/ml or higher which reflects the observation seen in the microscopic images.
GFP expression in HEK293T cells transfected with PS10025 plasmid DNA (1ug DNA/well of a 6-well plate) and treated with varied doxycycline (Dox) concentration for 2 days (original magnification, 10x)
A 96 well plate fluorescence reader was used to assess the induction of the Tet-ON system by Dox by measuring the GFP expression at 0, 24, 48, and 72 hours post induction. Cells were treated with 0, 0.01 0.1, 1, 10 (ug/ml) of Dox. GFP intensity readout from the plate reader was plotted on the y-axis (arbitrary units, a.u.) against cells treated with varying concentration of Dox on the x-axis at different time points. Maximal induction of our Tet-ON system (PS100125) was observed at 72 hours post Dox treatment for all doses of Dox whereas virtually none was quantified in cells that was not induced by the Dox
Dose response induction of Tet-ON system( CAT #PS100125) with Dox as measured by the influence intensity of GFP
Effects of doxycycline on expression of TurboGFP protein by Western blotting analysis. HEK293T cells were transfected PS100125 plasmid DNA (1 µg DNA/well of a 6-well plate) and treated with increasing doxycycline (Dox) concentrations (0, 1, 10, 100, and 1,000 ng/mL) for 72 hrs. Untransfected (UT) and transfected cells were lysed and analyzed using Western blot with TurboGFP-specific antibody (TA150075, ORIGENE). 25 μg of proteins were loaded on each lane. Anti-β-Actin mouse mAb (TA811000, ORIGENE) was used as loading control for Western blot. Bar graphs represent relative TurboGFP expression normalized with β-Actin.
Gossen, M. & Bujard, H. (1995) Efficacy of tetracycline-controlled gene expression is influenced by cell type. BioTechniques 89:213-215
|Inducible Vector Resources|