All-In-One inducible expression system

What are Inducible Expression Vectors?

Inducible Expression Vectors allow for spatial and temporal control of gene expression in mammalian cells. 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. There are two different types of inducible vectors:

• Traditional Inducible Expression Vectors
• Lentiviral Inducible Expression Vectors

Features of Tet-On system

• Presence of the transactivator and the promoter in the same vector, eliminating the need for another plasmid.
• Modified 3G transactivator for high doxycycline sensitivity.
• High level of induction with minimal background expression.

Fig 1 : FP expression in HEK293T cells transfected with plasmid DNA (1ug DNA/well of a 6-well plate) and treated with varied doxycycline (Dox) concentration for 2 days (original magnification, 10x)

Experiment : 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.

Conclusion: Raw fluorescence intensity readout (expressed in arbitrary units) in Fig 1 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.

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. 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.

Traditional Inducible Expression Vectors

Vector	E-coli Selection	Mammalian Selection	C-Tag	Promoter
pEntry-rtTA	Ampicillin	Neomycin	Myc-DDK	CMV
pCMV6-AC-GFP-rtTA	Ampicillin	Neomycin	tGFP	CMV
pCMV6-AC-tGFP-rtTA-Hyg	Ampicillin	Hygromycin	tGFP	CMV
pCMV6-AC-Myc-DDK-rtTA-Hyg	Ampicillin	Hygromycin	Myc-DDK	CMV

Table 1. OriGene's inducible systems

Fig 2 Dose response of Tet-On system with Dox as measured by the influence intensity of GFP

Fig 3.Effects of doxycycline on expression of TurboGFP protein by Western blotting analysis. HEK293T cells were transfected with 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.

References

Gossen, M. & Bujard, H. (1995) Efficacy of tetracycline-controlled gene expression is influenced by cell type. BioTechniques 89:213-215

Inducible Lentiviral Expression Particles (Overexpression and shRNA available)

(Exclusively Available through our Custom Lentiviral Packaging Services)

Vector	Mammalian Selection	C-Tag	Promoter
pLenti-Tet-On-GFP	None	tGFP	PGK
pLenti-Tet-On-Myc-DDK-Puro	Puromycin	Myc-DDK	PGK
pLenti-Tet-On-shRNA-Puro	Puromycin	None	PGK

Table 2. OriGene's Inducible Lentiviral System

Inducible Lentiviral Expression Protocol

Inducible Lentiviral Particles for Overexpression

Fig. 1. Dose-dependent GFP expression in HEK293T cells transduced with inducible lentiviral GFP particles and treated with varied doxycycline (Dox) concentrations for 5 days (original magnification, 10x). High GFP expression was observed by the treatment of 10 µg/mL Dox.

Inducible Lentiviral Particles for Gene Knockdown

Plasmid Maps