Greetings - I hope someone can help me understand this odd phenomenon!
When an integrated transgenic array (a translational fusion::GFP) marked with roller is crossed with different mutants, the resulting doubles will, in some instances, fail to roll. These animals are not dpy, but seem to move in a wt fashion. Why would this happen with some mutants, and not others?? More importantly, is it meaningful? By eye, the level of GFP expressed off the transgene does not seem effected, although I have thought it may be worth checking mRNA levels.
Any insight would be appreciated! Many thanks in advance…
If you mean for certain mutants, all of the progenies with the mutant won’t roll (instead of some roll but some don’t), my best guess is that those mutants are on the same chromosome as your roller gene. It is possible to get double mutant within the same chromosome, just the chance is lower than normal. Hope it helps.
The chromosome the transgene+rol is integrated on is known, and that does not track with which mutants suppress the roller phenotype and which do not (I’ve crossed this strain to many mutants). I also don’t quite understand why being on the same chromosome would affect the roller phenotype. Could you explain further? And yes, all progeny fail to roll for seemingly all subsequent generations, while they maintain GFP expression.
When I saw you saying “The chromosome the transgene+rol is integrated on is known” I realized your GFP and roller marker are integrated together, instead of integrate a GFP transgene on a roller mutant background… so what I mentioned earlier was not relevant. It actually says that rol-6 is “suppressed in some mutant backgrounds (unc, dpy)” (http://wormbook.org/chapters/www_transformationmicroinjection/transformationmicroinjection.html#d0e102). Although I am not sure which rolling marker you are using, this might be a similar effect.
To explain “why being on the same chromosome would affect the roller phenotype”: what I meant was, when they are on different chromosomes (RR on chromosome II and mutant MM on chromosome V for example), when crossing RRmm (roller) with rrMM (your mutant), you get 100% RrMm F1 worms, and 25% RRMM double mutant for F2 worms.
But if your roller phenotype marker is on chromosome II (RR for roller, rr for wild type), and the mutant to cross with is on chromosome II as well (MM for mutant, mm for wild type), then when you cross your strain (Rm)(Rm) (roller) with (rM)(rM) (mutant), it is less chance to get homozygous of (RM)(RM). The rate will be low and depends on chromosome recombination. The closer the genes are on the chromosome, the less chance they get crossover.
I hope these make sense.
Back when I worked with rol-6(d) co-injection marker a lot, I found the Rol phenotype to be enormously variable. And I observed different penetrance in different backgrounds. So, not too surprising.
One thing stuck in my brain, because we used it: mutations in sqt-1 (or RNAi) can suppress the Rol phenotype of rol-6(d) transgenes. This is useful if your Rol transgene needs to be non-Rol for you to score it. Not great, because the penetrance is just reduced rather than complete suprpression. But workable.