A single-vector tetracycline construct allows doxycycline-regulated expression. (A) Overview showing the regulator plasmid containing the EF1α-tTA cassette (pEntL1L3-EF1α-tTA, left) and the responder plasmid containing the TetO-mCh-Rs1 cassette (pEntR3L2 TetO-mCh-Rs1, right). The mCherry and Rs1 cistrons are separated by a P2A ribosomal skip sequence to allow simultaneous expression of both peptides. The entry plasmids were recombined using Gateway technology into the desired destination vector containing the AttR1 and AttR2 Gateway sites. The TetO and EF1α-tTA portions are in opposite orientation (indicated by upside-down text) to minimize steric hindrance between the two promoters, as well as potential cross-activation of the TetO by the EF1α promoter. In addition, flanking insulator sequences are included to minimize any read-through activation of the constructs by surrounding promoters (such as Rosa26) that may lead to "leakiness" or steric interference from endogenous promoter activity. (B, C) HEK-293 cells carrying the Exp-pcDNA3.2(EF1α-tTA/TetO-mCh-Rs1) expression cassette and cultured in doxycycline (suppressed expression) or in the absence of doxycycline (transgene expression allowed) demonstrate doxycycline-dependent mCherry expression. (D) Schematic of targeted Rosa26 locus and Southern screening strategy. The Rosa26 locus in E14 ES cells was targeted by homologous recombination with the Exp-R26(EF1α-tTA/TetO-mCh-Rs1) construct. Regions in hatch marks indicate the 5' and 3' homology regions of the targeting vector and the endogenous Rosa26 locus (abbreviated R26 in the figure). The location of the 5' recombination Southern probe and HindIII restriction sites are indicated. (E) Southern blots of genomic DNA digested with HindIII and probed as in (D). Heterozygous ES cells at the Rosa26 locus are indicated by the two bands.