Conditional protein stabilization via the small molecules Shld-1 and rapamycin increases the signal-to-noise ratio with tet-inducible gene expression

Cellular mechanisms control one or more of the three basic levels of regulation (transcription, translation, and protein activity/locality), allowing for finely tuned spatial and temporal regulation of protein expression patterns. Gene regulation constructs in wide use today often employ a constitutively expressed transcription factor whose activity is determined by the presence or absence of a small molecule. A case in point is the tet transcription system, wherein transcription is regulated by doxycycline (Dox), allowing the researcher to turn protein expression on or off, depending on the presence/absence of Dox. However, in many applications of that system, there is basal transcription from the promoter element that is independent of Dox. Moreover in vivo, heterogeneous distribution of Dox leads to concurrent differences in gene expression. We addressed these limitations by introducing conditional destabilizing elements to the system. First, we created a transactivator protein fusion regulated at the additional level of protein stability This modification enabled a system that demonstrated an off state that is less sensitive to variations in Dox concentrations. We also regulated the stability of the protein expressed from the tet operator cassette, observing greatly improved signal-to-noise ratios. The results underscore how investigator-defined regulation at multiple levels of protein expression can attain a finer degree of control over the final expression of introduced genes.