I’m injecting a GFP reporter along with the rol-6 coinjection marker to be expressed in N2 worms. I can get a fair number of rollers in the F1 generation (more than 50 and some of these are green), but I never see any rollers in the F2 generation. Right now I’m injecting the GFP transgene and rol-6 at 30ng/ul each and supplementing this with pBluescript vector to bring the overall DNA concentration to greater than 100ng/ul (I heard this can help heritability of the transgene). Any help would be appreciated!
My understanding from others is that rol-6 (pRF4) should be injected at higher concentrations, possibly up to 100 ng/ul on its own. I don’t think it should be necessary to supplement with additional DNA other than your GFP beyond that.
We typically pick 100 F1’s to get ≥5-10 stably transmitting F2s. I have found with two markers (one being GFP) picking F1’s that are rescued for both (roller and GFP+ in your case), you don’t have to pick as many F1’s. But you still should. The GFP+ guys should have GFP in most or all of the cells that you expect it to be in. For example, when injecting with myo-3::GFP, it should be in almost all of the body and vulval muscle cells to be considered GFP positive, not just a few muscle quadrants near the head. These animals tend to pass the array to the F2 progeny much more reliably than others.
i usually coinject the rol6 and gfp plasmid, the total DNA concentration is 100ng/ul, but i do not add any others plasmid. For example, when the gfp is 20ng/ul, the rol6 is 80ng/ul. rol6 is alway more than gfp.
when picking 20F1’s roller worms, i usually can get >=1 stably F2s.