Tet-On 3G Inducible Expression System (EF1alpha Version)

Description

The Tet-On 3G Inducible Expression System is a powerful, tightly regulated, tetracycline-inducible mammalian expression system that is controlled by the addition of doxycycline to the culture medium. In the EF1alpha Version, Tet-On 3G transactivator expression is driven by a human elongation factor 1 alpha EF1alpha promoter, which allows expression of the protein in a variety of mammalian cells such as hematopoietic or stem cells without the transgene silencing associated with CMV promoters. The system includes a vector set, our highly efficient transfection reagent, Xfect,and Tet System Approved FBS. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back The Tet-On 3G system is a highly sensitive tetracycline inducible expression system.Following cotransient transfection of pCMV-Tet3G and pTRE3G-Luc in HeLa cells, increasing levels of Dox were added and expression of luciferase was measured using an anti-luciferase antibody Panel A and a luciferase assay Panel B . Induced expression was very high even with Dox concentrations as low as 10 ng/ml, and was detectable even at 0.1 ng/ml Dox. Back Tet-On 3G results in the lowest background and as a result provides a much higher fold-induction than previous generations of the Tet-On tetracycline-inducible expression system. Back The Tet-On 3G system demonstrates higher sensitivity to doxycycline than Tet-On Advanced system.Tet-On 3G and Tet-On Advanced genes were integrated at the same locus in a stable HLF33 cell line expressing luciferase from a TRE promoter. For each of these two double-stable cell lines, induced luciferase expression was measured in response to a range of doxycycline Dox concentrations. At 510 ng/ml Dox, induced expression was 100 to 150-fold higher for the Tet-On 3G cell line, and at 50 ng/ml, expression was 4.6-fold higher data kindly provided by Professor W. Hillen, and Dr. C. Berens, University of Erlangen . . Back ThePTRE3Gpromoter results in significantly reduced basal expression.HEK 293 cells were transiently cotranstected with both the response vectors containing luciferase and regulator vectors from each of the Tet-On 3G and Tet-On advanced inducible expression systems. The cells were cultured in the presence and absence of Dox, and after 24 hr, luciferase expression was measured. Although both systems provided strong expression in the presence of Dox, the Tet-On 3G system produces far lower background expression in the absence of Dox inset . Back Back Tet-On 3G inducible expression systems with EF1-alpha regulator vectors. Required Products Fetal Bovine Serum FBS that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline or derivatives which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems. This FBS has been processed in the United States. Back Fetal bovine serum FBS approved for use with tetracycline inducible expression systems.Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels. Back Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems.Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS. Back Fetal Bovine Serum FBS that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline or its derivatives which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems. Back Fetal bovine serum FBS approved for use with tetracycline inducible expression systems.Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels. Back Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems.Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS. Back