Was hoping someone could help me answer a question I had…
I have generated a GFP-reporter for a gene and coinjected it into N2 worms with the pRF4 plasmid( rol-6).
I’ve just seen however, that the gene is more predominantly expressed in males and was wondering if it would be possible to get males
from my already injected N2 worms without having to inject the construct into a him stain? Also, how would I be able to identify the roller males? would
their phenotype make them harder to distinguish from females?
Getting the males is easy enough: heat-shock (or ethanol-shock) the transgenic line for males, or use the same method to make a mating stock of N2 and mate the N2 males you get from that stock to transgenic hermaphrodites. It shouldn’t be hard to identify the rollers that are males. If you haven’t made males before, there are some protocols in this thread.
On the other hand, keeping the males is more likely to be problematic. Roller males don’t mate well (though I’ve heard that if you’re sufficiently determined you can get it to happen), and so once you have the transgenic males you probably won’t be able to maintain a mating stock to have a ready source of more transgenic males - though it’s easy enough to keep a mating stock of N2, and mate those males into your transgenic line whenever you like, or to mate non-Roller male progeny to Roller hermaphrodite progeny. Still, this is one of the reasons a lot of people don’t recommend using Rol as your co-injection marker (that the whole animal is twisted will also be inconvenient for figuring out your expression pattern and for getting usable images).
I’ll echo Hillel here. rol-6(d) males stink; the male tail fan is strongly cupped, which I assume is what compromises mating. Though I have gotten them to mate, you really have to brute force it.
And I did mine from a strain that had him-8(e1489), so lots of available males. Doing so by heat shock would probably be prohibitively difficult. So, rol-6(d) (pRF4) is something I’m not using anymore.
If you must use it, consider a trick. sqt-1 RNAi can diminish the Rolling. This helps with GFP visualization, but I can’t say what it would do to mating.
It probably depends on how strong the roller phenotype is in the particular line. Sometimes the cross will work reasonably well, but if the males are strong rollers they will just make a “C” and spin most of the time. Alternatively, you could just take your roller hermaphrodites and let them run out of sperm (takes 2-3 days after reaching adult at 20 degrees) and then mate the “females” with males from your other stock. I do like the idea of civilizing the males by RNAi. Seems like RNAi of rol-6 should work too since rol-6(0) is wild type.
Thank you all for your input, I’ve considered using non-roller males now. Thank you