Hi everyone,
I am new to C. elegans research, so I’m hoping y’all can help. I am doing feeding RNAi to look at the genetics of cellular defenses against ingested pathogenic bacteria.
Since the strain I am using, VP303, only performs RNAi in the intestine, my usual unc-22 control to verify the RNAi effect doesn’t work. Furthermore, rather than growing the worms from the L1 stage on RNAi bacteria, we decided to grow the worms on normal OP50 till they are L4s and then do the RNAi (for 2 days) before exposing the worms to the pathogen, to avoid the possibility that any effect on worm survival we see after knockdown may simply be due to loss of the targeted gene screwing up intestinal development.
So my question is, does anybody know of an Ahringer or Vidal library clone that can be fed to L4 worms, acts at the intestine, and gives an easily scored phenotype that I can use to quickly verify that knockdown successfully occurred?
Thanks!
Do pmk-1 (rnai) or sek-1 (rnai) as you describe and look at resistance to P. aeruginosa. I’ve done this many times and they die faster than wild-type in an obvious way.
I’ve never used them, and in particular don’t know whether they work well by RNAi, but you might try the glo genes, which in theory you ought to be able to test directly on the RNAi feeding plate using a dissecting scope with fluorescence optics, or by picking some worms off and looking by DIC. Still maybe not a great choice (especially considering the disclaimer about not knowing whether they will work by RNAi), but could be less work than doing a pathogen-sensitivity assay.
You could cross that strain with a strain that carries a gut specific GFP reporter (ie elt-2::GFP, pho-1::GFP, pep-2::GFP) or a GFP reporter that is expressed in all cells (ie histone GFP) then do RNAi against GFP and show decreased/absent GFP expression in the gut.
This does require making a new strain, but it would give you a very easily scored result.