We’re having an RNAi efficacy issue that’s turning into a real head-scratcher.
We’ve been testing worm strains for RNAi sensitivity by the following protocol: transfer L4s to RNAi plates, eventually transfer individual adults to single plates, remove, count proportion of embryos that hatch. On embryonic lethal vectors, we see 100% lethality in sensitive strains (like N2).
However, as other labs have noticed, the earliest-laid eggs do hatch on embryonic lethal vectors (but they are not part of the assay, which examines hatching on the single plates). In an attempt to get 100% lethality across ALL embryos, we have transferred worms as L1s onto the RNAi plates, so the egg-laying adults are exposed to RNAi over their whole lifetime. Still, we see that the earliest-laid eggs hatch. We see similar results in liquid culture.
We have mostly used par-1 as our embryonic lethal vector but we are trying others. One thought was that in order to induce the RNAi response, the target gene must be sufficiently expressed. Since we are targeting embryonic genes, perhaps there is a lag time between the first time the gene is expressed, and the full effect of the RNAi response… consequently, the earliest-formed embryos may scoot through development (and hatch successfully) before they can be effectively interfered with. However, par-1 is expressed in the larval and adult stages, so there should be a systemic RNAi response before those eggs are even formed, right?
Any ideas why RNAi does not seem to be affecting the earliest embryos?
Does anyone know of an embryonic lethal vector that does give true 100% lethality… or a modification to the protocol that will yield 100% lethality?
Eventually we want to get this to work in liquid culture, for what it’s worth.
i suggest inducing RNAi generation with IPTG prior to plating the bacteria then 24 hours after spotting the plates with pre-induced bacteria put down L1s. par-1 is a maternal effect lethal so if you don’t knock it down completely by adulthood the early embryos will be rescued but be infertile.
Hi, thanks for this, this sounds like a good thing to try.
We have kept track of how long the bacteria have sat on the plates before doing the assay, and found no difference among plates that were seeded with bacteria between one day and one week before use. We haven’t induced with IPTG before plating, however. We do induce the bacteria in liquid culture before adding the worms (by a few hours). If anything we see more hatching in liquid culture. It may be worth inducing the bacteria for a lot longer in liquid culture as well.
When you grow the overnight culture, the antibiotics can wear out, allowing bugs without necessary vectors to grow in your culture and reducing RNAi effect. If you want to be extra careful, you should dilute the overnight culture and grow cells to log phase, then induce at room temperature for a few hours- similar to how you treat bacteria when inducing them to produce proteins (See: http://www.ncbi.nlm.nih.gov/pubmed/11223248). Also, we have found that growing bacteria from -80C stocks on amp/tet plates can be very important. We noticed that if the amp/tet plates used for growing from -80C aren’t good, then RNAi effectiveness drops even though you subsequently grow the bacteria in liquid culture with selection.
So, use fresh amp/tet selection for each step of culture, make sure the bacteria aren’t more than two weeks old, and try using ideal culture conditions before inducing. As for RNAi in liquid culture, I have the impression that it doesn’t work. If anyone has made it work I would also like to know the protocol!
We find this protocol to work quite well. The question I’m struggling with now is why RNAi does not seem to affect ALL individuals, when biologically I can’t think of a good reason for it. Specifically, in the case of embryonic lethal vectors, why the earliest-laid embryos appear (partially) insensitive to RNAi and go on to hatch (though they don’t develop normally).
I think this phenomenon might be a fairly common and generalized one, because I’ve talked with other researchers who have optimized RNAi in liquid culture for very large-scale experiments, and they expect to see “escapees” in the wells. They just work around it by establishing a baseline phenotype to compare everything to. And like I mentioned above, we see this phenomenon on solid culture as well.