Figure 1.
Engineering an in vivo system for conditional overexpression of genes in the megakaryocyte/platelet lineage. The scheme shown illustrates the Tet-on/off inducible gene expression system, involving the interaction between 2 transgenes of interest. The tissue-specific PF4 promoter drives the expression of the transactivator tTA (fused to the enhancing factor VP16). This line is crossbred to a tetracycline responsive element (TRE) bidirectional CMV line. The use of the TRE bidirectional CMV promoter allows the simultaneous regulation of both Aurora-B and β-gal by one central TRE. The TRE is a stretch of 7 copies of the 42-bp Tet operator sequences that bind to tTA-VP16 fusion protein in the absence of tetracycline/doxycycline. To achieve inducible transcription of 2 transgenes, the TRE is sandwiched between 2 minimal CMV promoters (directing the transcription of Aurora-B and β-gal in opposite directions, presumably in the absence of tetracycline).