Hi everyone,
I’m planing to measure life span of integrated transgenic animals.
Since the transgenic animals are roller, I want to use roller worms as a control.
Which strain do you use for roller control?
Can I request it from CGC?
Thanks.
That could be problematic. You would want something as close to isogenic to your transgenic strain as possible. However, if you have only a single integration line, then any changes you see could be due to disruption of the chromosome by the integration event. Procedurally, the simplest thing to do would be to take the original strain that has the extrachromosomal array from which the integration was derived and a) isolate multiple independent integration lines, and b) take non-Rol animals from the original strain, inject them with pRF4 to get worms carrying arrays, then isolate multiple independent integrated lines for these. Then backcross each strain about 6X using the appropriate background strain (N2 or whatever you are using). To do it properly with rol-6 is going to require quite a bit of work, so you might want to check and see if the aging community prefers some other marker for transgenic comparisons. You can always do a quick and dirty comparison just to see if the integrated line that you have is obviously different from wild type (or a simple pRF4 integrant); however, I’m not sure where you go from there since it is not a publishable negative result and a positive result is going to require proper controls in order to be credible.
The Reagents part of the WormBase page for rol-6 names an integrated transgene, hIs1, that apparently contains only rol-6(su1006); you could start with that. I thought I remembered another one, but a quick glance at the papers I thought might have it yielded no indications my recollection was right.
The good thing about hIs1 is that (according to WormBase) it was a spontaneous integration event, so at least there shouldn’t be all that many mutations hanging around from the gamma, x-rays, or other agents usually used to induce transgene integration. And everyone uses basically the same rol-6 transgene, so that will be consistent. But they might not have used the same amount of transgene, which may well matter, and as lmu1 stresses in their response the differing strain background in which they obtained their integration can make a big difference. See Patel et al for a great example of this, one that has obvious relevance to your situation. And note that in this paper both of the differing strains were called “N2”; you can’t trust that a transgene outcrossed by another lab to N2 has been outcrossed to the same N2 you use - though getting your N2 from the CGC, freezing many copies quite soon after receipt, and thawing regularly will help.
Your best bet in terms of a good-faith but limited effort to do the control may be to outcross hIs1 to your wild-type strain several times - though this necessarily won’t replace any possible functional polymorphisms near the hIs1 integration site, and it’s not uncommon for integration events to involve genomic rearrangements that suppress recombination across a large part of the chromosome. lmu1’s suggestion that you make several integrants of a rol-6(su1006)-only transgene, outcross each, and use them as several controls is definitely the most rigorous approach, but is a significant amount of work.
Another answer, not terribly helpful, is that rol-6 is not a marker I’d recommend for just this reason, because it makes the worms abnormal - although I suppose that in theory you might be obligated to do the same test for any other coinjection marker you might choose, even those that superficially appear to rescue to wild-type. Still, if you can move away from rol-6 for such experiments going forward, that would seem desirable.