Hello,
I am trying to activate a pro-longevity factor at different time points after adulthood to define its action window to extend lifespan throughout its life. It seems that a variety of heat-shock promoters is commonly used. But I am not really sure which of these promoters can still be induced by heatshock along the entire adulthood. Any suggestions to the promoters or strategy to study this question would be very helpful. Thank you very much.
Hi,
just to get the ball rolling here, as you posed a question and (at least) 85 people have not come forward with anything.
As a way of making people reply, I’ll give you a theoretical viewpoint (I haven’t used heat shock promoters in the way you describe), which should bring out the experimentalists to give me a kicking and you some advice.
Two papers, both alike in dignity (to misquote Shakespeare) and in the public domain:
http://www.impactaging.com/papers/v1/n6/pdf/100058.pdf
http://genesdev.cshlp.org/content/12/13/1962.full.pdf+html
The former reviews the role of heat shock proteins in mice and worms…reiterates that there seems to be a clear longevity effect (a plus point for your research), but also that this effect is complex (still a plus point for your research).
What I find interesting is the idea that different tissue/organ systems exhibit differential responses to heat shock (in terms of extending lifespan), it is not all on the +ve side.
That leads me to the latter of the two papers. It’s from Dr. Heat Shock’s group (Richard Morimoto) and considers the role of a heat shock binding protein that negatively regulates heat shock protein binding (strange eh?). Anyway, over-expressing it off a tissue specific (muscle) promoter in worms shuts down a heat shock-inducible construct hsp16::lacZ in the same tissue but not in other tissues where it is normally expressed.
Thus, (and here’s the bit where people start getting itchy fingers to write NNOOOOOOOO), I wonder if you might have to ‘just do the time’ and test the expression of your heat shock promoter constructs tissue for tissue over the adult period? It could be, that some tissues express HSBP-1 and effectively block the heat shock response in older worms in certain tissues but not others beacuse it’s advantageous to do so?
One example, drawn admittedly from work with mice, is that the effects of heat shock proteins on longevity seem to be more pronounced in certain tissues such as cardiac tissue than in others.
steve
What about starting with hsp16 (nerve ring, pharynx, intestine, body wall muscles)?
Steve, I fear you’ve misunderstood. AngelaL is proposing to use heat shock as an inducible overexpression system purely as a tool to study how overexpression of some gene of interest affects lifespan - not necessarily to study the contribution of endogenous heat shock response to lifespan.
Indeed, this is why I’ve hesitated to respond: because it’s already well known that transient heat shocks will extend animal lifespan, as will other stresses such as alcohol exposure - presumably through activation of stress-response pathways known to be deeply involved in determining lifespan. This makes the whole project somewhat problematic. Sure, you can do the appropriate controls, but those controls are likely to already include lifespan extension, making interpretation hard in a couple of ways. Maybe there’s a mutation that will abolish stress-induced lifespan extension (I haven’t checked the literature), but this might also block a real effect of AngelaL’s transgene.
If I had a good idea for a tested inducible promoter that didn’t involve causing animal stress, I’d mention it - but I don’t have one. I can imagine ways of making one (for example: move animals onto RNAi bacteria targeting a transcriptional repressor that is preventing expression of your transgene), but I don’t know of one that’s tested and ready.
The short answer is simple: I don’t know which heat-shock reporters are inducible in adults, but it’s easy enough to find out, by testing heat-shock::gfp transgenes. But, as the papers you cite indicate (and even more so other papers in elegans that I haven’t bothered to look up), heat shock may not be the answer.
you see…it had an immediate effect, gladly the kicking was given with carpet slippers though, thanks Hillel!
Yep, perhaps I was too much into the differential effects of heat shock to see the ‘means to an end’ wood from the ‘heat shock response’ trees. I guess I had in mind that if AngelaL’s gene was tissue-specific then it’s effect might not be seen after heat shock because in that tissue it would be repressed by HSBP-1…
But Hillel is of course correct, using heat shock inducibility to study the effects of a GOI on lifespan is problematic given the known effects of heat shock on longevity.
Using opto-inducible constructs is another possibility, but then as far as I know, most would cause a concurrent heat shock/stress response…and wavelengths that don’t (around 800nm for example) are not available.
Or, using stage-specific promoters (with their limitations) coupled with your GOI that could (crudely) delineate your adult into developmental periods? You would have to look these up, but two examples, the vitellogenin promoter (late L4/adult), col-19 (L4/adult).
Steve
Although a lot more work, one could consider setting up the Q system: http://www.ncbi.nlm.nih.gov/pubmed/22406855
It’s a binary system similar to Gal4-UAS. You could put the gene of interest of interest under the control of the QUAS response element, and then choose a promoter that expresses in adults. Maybe mine some modENCODE data (I’m sure that there must be some older animal RNA-seq data) to find a good promoter. Then put the transcriptional activator (QF) and the transcriptional repressor (QS) under control of this promoter. Additional of quinic acid will alleviate repression and allow induction of the transgene. You’d have to see if quinic acid alone affects lifespan, but this may circumvent some of the stress response issues used by other inducible reporters, nicely highlighted by Steve and Hillel. One other consideration is that it may be worth putting a fluorescent reporter either on your gene or separated by a viral 2A sequence, so that you can monitor induction and get an idea of kinetics.
Okay. Thanks all. I really appreciate that. 