Conditional expression of mPer2 in NIH 3T3 fibroblasts using the Tet-Off system. (A) Schematic drawing of the tetracycline-regulatable mPer2 expression system. Dox represents doxycycline, which is a derivative of tetracycline. In this Tet-Off system (BD Biosciences Clontech), the tetracycline-controlled transactivator (tTA), which is composed of tetracycline repressor protein (tetR) and the VP16 activation domain of herpes simplex virus, is expressed from the constitutive cytomegalovirus promoter (CMV pro). When Dox is absent (Dox−), tTa binds to TetO (tetracycline operator) and Pmin (CMV minimum promoter) activates mPer2 expression. In the presence of Dox (Dox+), mPer2 expression is not activated (B) Expression of exogenous mPer2 mRNA in pTet-Off and pTRE2-mPer2 double stable NIH 3T3 cell lines. The P2-#8 cell line shows almost no expression of mPer2 under Dox+ conditions, whereas apparent induction of mPer2 is seen under Dox− conditions. P2-#19, P2-#21, and P2-#22 cell lines represent weak basal expression under Dox+ conditions and strong induction of mPer2 under Dox− conditions.