Can the Clontech Tet-On® system or Tet-Off® system components be used with your T-REx™ tetracycline-regulated mammalian expression system?

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Answer

No. The two systems are not compatible since they utilize different strategies for promoter regulation. The T-REx™ system is designed such that native E. coli tet-repressor protein molecules bind to specific tet-operator sequences (2X TO) just downstream of the TATA box in the full length CMV promoter in the expression vector. This binding keeps the promoter silent simply by preventing the normal transcription machinery from productive assembly at the TATA box. Incidentally, it is this full length CMV promoter region that permits higher induced expression levels relative to other systems.

The recombinant ’repressor’ proteins utilized in Clontech’s system are actually recombinant fusion proteins which also contain a potent transcriptional transactivator. The Clontech system places operator sequences 5’ to the TATA box and relies upon the VP16 transactivator to promote transcription. These repressor-transactivator fusion constructs would have unpredictable and unreliable effects at the CMV promoter in our expression constructs. Additionally, the tet-repressor protein produced from the pCDNA™6/TR construct in the T-REx™ system has no transactivation domain and so would exert little regulatory effect at the minimal promoter region (non-full length CMV) found in the Clontech response plasmids.

Answer Id: E3947

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