over a month ago the lab prepped a new stock of pRF4. and since then i have been having problems getting lines. i get plenty of transgenic F1’s but very very few lines. the last injection, i had 250 F1 rollers and only 1 line. previously, i injected different constructs at normal concentrations (5 to 10ng/ul of the construct along with 100ng/ul of pRF4). and, for each injection i single out over 100 F1 rollers and only get 1 or 2 lines from them. all the plasmids are different (different promoters and different genes). it is highly unusual, since before the new pRF4 stock, i would get approximately 10 lines from 100 transgenic F1. i suspect it could be the pRF4. fortunately, there is still an aliquote of some old pRF4 (which transmits just fine), so i can do a control. but until then, anyone run into said issue?
we’ve had similar troubles before, in a slightly different context - pha-1 rescue. We’d inject, get 40-50 F1s, none of which transmit (compared with a ~10-20% transmission rate in “normal” conditions). We worked around that annoying problem by having a frozen stock of pha-1 that we’d thaw afresh every month or two. Maybe you could try to inject in a freshly thawed N2 stock.
few months ago I also did some microinjections of promoter::GFP constructs using pRF4 and rab3::cherry as co-injection markers…and I had the same problems with roller animals.
However I always could detect cherry fluorescent animals also expressing GFP from the transgenic construct…
I think it could be a general problem for pRF4 marker because animals are still keeping the transgenic construct even after “loosing” the roller expression.
Are you co-injecting your constructs only with pRF4?
Maybe you can try to co inject your animals together with pRF4 and another marker and check if this is also happening in your case… or directly use another co-injection marker alone…
Good luck !!
pRF4 can be fickle. Often enough I’ve gotten weak rollers, where all they do is lift their noses. I pretty much avoid rol-6 these days, preferring to go with gfp markers.
But sounds like Dan, the original poster, might be having a DNA prep issue and not a specific marker issue.