pTet-Off Advanced Vector

Description

The pTet-Off Advanced Vector is used to create stable Tet-Off Advanced cell lines as hosts for a Tet-inducible expression system. The Tet-Off Advanced protein is a modified Tet-responsive transactivator tTA-Advanced that binds tightly to tetracycline-responsive elements TRE and strongly activates transcription of a downstream gene in the absence of tetracycline Tc or doxycycline Dox; a Tc derivative . As Tc or Dox is added to the culture medium, transcription from the TRE or TRE-Tight is turned off in a highly dose-dependent manner. The Tet-Off Advanced gene is fully synthetic and has been optimized for expression and stability in mammalian cells by incorporating mammalian codon preferences and by removing cryptic splice sites from the mRNA. In addition, 3 minimal F-type HSV VP16 transcription activation domains provide maximal activation and reduced toxicity. The immediate early promoter of cytomegalovirus PCMV IE produces strong constitutive expression of Tet-Off Advanced, which accumulates to high levels. Stable Tet-Off Advanced cell lines can be generated by transfecting the plasmid and selecting for G418-resistant clones. 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 Induced expression in the Tet-Off Advanced and Tet-On Advanced systems.The Tet-controlled transactivators are fusion proteins that contain a DNA-binding TetR domain joined to three minimal transcription activation domains from HSV VP16. Each transactivator has been optimized for expression in mammalian cells. In Tet-Off Advanced systems, the basal state is maintained in the presence of doxycycline Dox , and induced by its withdrawal. Tet-On Advanced systems are activated in the presence of Dox. System induction produces high-level transcription of your gene fromPTight. Back Induced luciferase expression in Tet-On Advanced and Tet-Off Advanced Expression systems.In the absence of Dox, basal expression was undetectable in Tet-On Advanced cells, whereas fully induced expression in the presence of Dox was very high. Tet-Off Advanced cells show the opposite response to Dox, with extremely high fold-induction, and undetectable basal expression in the presence of Dox. Back Tet-On Advanced systems are more sensitive to doxycycline than the original Tet-On systems.HEK 293 Tet-On and Tet-On Advanced cell lines were transiently transfected with TRE-luciferase vectors and treated with increasing concentrations of Dox. The Tet-On Advanced protein is tenfold more sensitive to Dox. Back