Him- feeding RNAi bugs

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I need males of a strain that’s got a ts mutation which disrupts cell division. So I heat-shock L4s, and search for males among their offspring. But the offspring are messed up! They are sluggish, and most are Pvl and Ste. I don’t know what’s going on, but suspect widespread genomic catastrophe, and I don’t want to use these guys in a cross. Alternative: do RNAi to knock down a him gene. Can somebody send me the feeding RNAi bugs for some him? I’ll be very grateful.

It would be very handy if there were an easy-to-RNAi gene that generated males without killing most progeny. The best choices would theoretically be him-8 or him-5, which selectively disrupt X-chromosome segregation, but neither is very amenable to RNAi. I would suggest trying klp-3 or klp-15, which are fairly sensitive to RNAi, although not X-chromosome-specific. Alternatively (and probably more simply), you could cross WT males to your strain to generate heterozygous males (and use those to create a mating strain, if desired).

It’s not a trivial task for someone to locate, thaw, and send a specific RNAi construct, but maybe someone out there has one that they use frequently that does generate males; I’m interested to know.

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Thanks for the quick response! I don’t want to cross this strain because it’s homozygous at 3 unlinked loci, and plates get expensive… I did not know that him-5 and -8 are specifically involved in X disjunction. That’s very interesting - I wonder if they get downregulated in situations where worms want to make more males.

I wonder about the basis for saying that him-5 and -8 are not very amenable to RNAi. I’ve just looked, and found two articles reporting effects of him-8 RNAi. Neither one mentioned the Him phenotype, but I guess it’s doing something… Even if him-8 is only a little bit sensitive to RNAi, that might be enough to get sufficient males to start the mating game?

If it works good enough, I think him-5 or -8 feeding RNAi should be a standard method for male production, instead of heat shock. Thermal stress is bad - I started wondering if it might be mutagenic, and did a quick search, and it is! This study Thermal stress accelerates Arabidopsis thaliana mutation rate reports doubling of point mutations and 20-fold increase of indels. That’s at a temperature where the organism (a eukaryote) is viable over many generations. So there seems good cause for concern… I have tried growing N2 at 26.5C, and they are fine; at 27.0C they go mostly Ste, and go extinct within a very few generations. Who knows what might get broken during four hours at 30C?

Well, it’s certainly worth a try to RNAi either him-8 or him-5 – either gene can be (partially) knocked down by injected dsRNA, but feeding is less effective, and you probably don’t want to inject just to get males on a routine basis, although in your particular case it may be worth it. Another gene you might try is him-1, which is not X-specific but preferentially affects the X (Meiotic mutants that cause a polar decrease in recombination on the X chromosome in Caenorhabditis elegans. | Genetics). It may also be worth cloning an RNAi construct into a vector that contains transcriptional terminators flanking the insert site, since this approach has been reported to be somewhat more efficient (Highly efficient RNAi and Cas9-based auto-cloning systems for C. elegans research | Nucleic Acids Research | Oxford Academic).

Actually, while looking for the T7 terminator reference I also turned up this paper, so you may want to contact the authors to get their “male-food” constructs: https://febs.onlinelibrary.wiley.com/doi/full/10.1016/j.febslet.2014.07.023

Heat-shock is almost certainly mutagenic - the Libuda lab recently published that heat induces DNA breaks due to transposon excision during spermatogenesis (Redirecting) so you’re right, it’s not a benign way to generate males. Moreover, heat shock is not terribly efficient, and the males that do arise after heat shock are often subfertile due to aneuploidy or other issues.

Good luck!

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Many thanks Abby Dernburg! And big thanks to Sturm et al., and to Timmons (again) et al. Wow, how awesome is the Worm Community Forum?

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PPPS. “Many labs use an RNAi construct that targets him-14 (GC363; available from the stock center).”

Fay D.S. Classical genetic methods (December 30, 2013), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.165.1, http://www.wormbook.org.