Tet-On 3G Bidirectional Inducible Expression System (EF1alpha, mCherry)

Description

The Tet-On 3G Inducible Expression System EF1alpha, mCherry is a powerful, tightly regulated, tetracycline-inducible mammalian expression system that is controlled by the addition of doxycycline to the culture medium. This system allows the simultaneous expression of a gene of interest and a red fluorescent protein mCherry marker from a bidirectional, Tet-responsive promoter that binds the Tet-On 3G transactivator protein in the presence of doxycycline. It includes the pTRE3G-BI-mCherry Vector Set, consisting of a bidirectional vector into which the gene of interest can be cloned and simultaneously expressed with the mCherry marker, a bidirectional control vector that contains the luciferase gene in one of the two possible cloning sites, and two linear selection markers for hygromycin and puromycin resistanceand the pEF1a-Tet3G Vector, which constitutively expresses the Tet-On 3G transactivator protein from a human elongation factor 1 alpha EF1alpha promoter, allowing expression of the protein in a variety of mammalian cells such as hematopoietic or stem cells without the transgene silencing associated with CMV promoters. This system also includes our highly efficient XfectTransfection Reagent 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 Tet-On 3G inducible expression systems with EF1-alpha regulator vectors. Back Doxycycline-regulated expression of two genes.ZsGreen1and firefly luciferase were cloned into separate multiple cloning sites that flank the inducible bidirectional promoter in the pTRE3G-BI vector. The plasmid was then cotransfected into HEK 293 cells with pCMV-Tet3G which expresses the Tet-On 3G transactivator . Cells were treated with the indicated doses of doxycycline, and induced gene expression was measured 24 hr later. Induced expression ofZsGreen1fluorescent protein was determined by fluorescent microscopy and luciferase activity was measured using a luminometer RLU = Relative Light Units . Back PTRE3G-BIinducible bidirectional promoter shows equivalent induced expression of two genes.ZsGreen1andmCherryfluorescent proteins were cloned into separate multiple cloning sites that flank the inducible bidirectional promoter in the pTRE3G-BI vector. The plasmid was then cotransfected into HEK 293 cells with pEF1a-Tet3G which expresses the Tet-On 3G transactivator . Cells were treated with 100 ng/ml doxycycline and induced expression of the fluorescent proteins was observed 24 hr later. Cells in the mixed population that expressed high levels ofZsGreen1also showed similarly high levels of mCherry expression, indicating equivalent expression from both sides of the bidirectional promoter. Back 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