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 afiner degree of control over the final expression of introduced genes.
Almogy
Gal
G
The Department of Microbiology and Immunology, Stanford University School of Medicine, Palo Alto, CA 94305, USA.
Nolan
Garry P
GP
D000818:Animals; D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis; D051379:Mice; D009025:Morpholines / Q000494:pharmacology*; D041681:NIH 3T3 Cells; D055550:Protein Stability / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D012097:Repressor Proteins / Q000378:metabolism; D020218:Response Elements; D020123:Sirolimus / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D009025:Morpholines; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C540198:Shield-1 compound; D015534:Trans-Activators; C485184:enhanced green fluorescent protein; C034811:tetracycline resistance-encoding transposon repressor protein; D049452:Green Fluorescent Proteins; D004318:Doxycycline; D020123:Sirolimus