We’re trying to express a toxic transgene that we’re normally keeping off by suppressing it with RNAi feeding that targets the transgene. To derepress the transgene we plan on transferring the worms to normal media but ideally we’d be able to shut off the RNAi response by transferring the worms to RNAI food that targets the RNAi machinery. I know the Mello lab used RNAi to identify things in the RNAi pathway but I don’t know that literature well enough to know which component of the RNAi machinery would be best to inhibit an already active RNAi response?
I haven’t used it and obviously can offer no guarantees, but have you looked into the Q system?
It’s not quite the same - it offers conditional relief from transcriptional repression, instead of conditional relief from post-transcriptional regulation - but it might work for you.
I had heard of the Q system, but I had also heard anecdotally that it wasn’t working particularly well for some people. Maybe that’s not generally true? If it works great then I’d absolutely give it a shot, even though it’s significantly more complex, but it seems like somebody must have isolated a TS mutant in the RNAi pathway that would allow one to do this via temperature shift?
Another way forward might be to bypass the transcriptional and post-transcriptional approaches and go to translational regulation. Stuart Kim and Tom Wandless’ groups described using destabilizing domains. These are amino acid tags derived from E. coli DHFR that result in protein degradation until one adds a stabilizing ligand (trimethoprim I think?). Looks like protein is stabilized at good amounts in 6 hours, reaching max levels in 12 hours or so. I had looked at this system a while back, but for our applications a degron was more useful. Only caveat is that I haven’t personally tried the destabilizing domains. They also describe a system based on FKBP, but I’d stay away from that one as the Shield-1 ligand was really expensive for very small amounts. http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0072393
There is also the Auxin-inducible degron (AID) system. The protein of interest is tagged with the AID, and that is expressed in a background expressing a TIR1 co-factor that targets the AID for degradation. So, you can rapidly degrade or restore expression of a tagged protein by addition/removal of auxin (a plant hormone). By restricting TIR1 expression to certain tissues, you could achieve tissue-specific regulation. Quite cute!
@Dave-yeah, it looks like a neat system! And I agree that the auxin degron could be another nice approach to achieve the same effect. Only reason I suggested the DHFR system is that it is a bit faster to restore protein expression. The auxin system takes ~24 hrs to restore maximum expression at 1mM auxin (can speed that up with lower auxin concentrations, but at the cost of slower depletion).