response plasmid that expresses a gene of interest (Gene X) inCLONTECH\'s Tet-On and Tet-Off Gene Expression Systems and Tet-On and Tet-Off Cell Lines (1). TheTet Expression Systems and Cell Lines give researchers ready accessto the tetracycline-regulated expression systems described byGossen Bujard (2; Tet-Off) andGossenet al. (3; Tet-On). pTRE2hyg contains anMCS immediately downstream of theTet-responsivePhCMV*-1promoter.cDNAs or genes inserted into the MCS will be responsive to the tTAand rtTA regulatory proteins in the Tet-Off and Tet-On systems,respectively.PhCMV*-1containsthe Tet response element (TRE), which consists of seven copies ofthe 19-bp tet operator sequence (tetO). The TRE element is justupstream of the minimal CMV promoter (Pmin CMV), whichlacks the enhancer that is part of the complete CMV promoter.Consequently,PhCMV*-1issilent in the absence of binding of TetR or rTetR tothetetOsequences. Note that thecloned insert must have an initiating ATG codon. In some cases,addition of a Kozak consensus ribosome binding site (4) may improveexpression levels; however, many cDNAs have been efficientlyexpressed in Tet systems without the addition of a Kozak sequence.pTRE2hyg also contains the hygromycin resistance gene for directselection of stable transformants. The parental vector pTRE2 wasoriginally described as pUHD10-3 in reference 5.

The pTRE2hyg-LucControl Vector, packaged with the pTRE2hyg Vector, contains anadditional 1649 bp encoding firefly luciferase inserted into theMCS. This vector can be used as a reporter of induction efficiencyusing standard luciferase detection reagents. It is not intended asa cloning vector.

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