Hi Forum,
I am working on an egg-laying assay to test a drug that acts on the NFY transcription factor, and among other things reduces the fertility of the worms. The drug works in the femtomolar concentration and has strong antimicrobial properties, and I am having some trouble deciding on how to treat/dose the worms with the drug. If anyone has any suggestions on good ways to treat the worms that would be extremely helpful.
Thanks
e.chang
Hi,
There a number of parameters that you can consider. I usually work through different permutations of these parameters to develop a sensitivity assay.
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Assay endpoint. You could look at animal development, embryonic lethality of the F1, or number of eggs laid by the treated animal. What end point you decide to use may affect your choice of the other parameters, and whether the protocol for DNA damage agent sensitivity assays that I’ve outlined is useful
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Liquid vs. solid media. I’ve seen some reports where the drug is put into MYOB agar, but if your drug is antimicrobial, then you might need to seed the plates with concentrated OP50, since the drug may interfere with bacterial growth. The cost of the drug and how much of it you have also affect this choice. With limited quantities of drug I prefer 96-well liquid assays to maximize the number of assays that I can perform. When I did DNA damage agent sensitivity assays, we picked animals into 6-well plates S-basal liquid media containing the drug and added concentrated HB101. The worms were incubated for 20 hours in the drug, then we transferred to an MYOB plate for several hours. In our hands, recovery on solid food was critical for good egg laying. Then we picked animals to single wells of a 24-well plate with MYOB, let the animals lay eggs for four hours. The worms were removed, the eggs counted, and dead eggs were counted 2 days later.
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Nature of the drug. ie. do you need to add any compounds to increase drug availability. In Ben Lehner’s puromycin selection protocol, addition of 0.1% Triton X-100 dramatically increased the sensitivity of animals without the resistance marker to the drug.
One consideration that you should make in interpreting any data that you get is that any effects of the drug could come from interaction with the drug with the E. coli, rather than the worm. I saw David Gems speak at a recent meeting where he presented data suggesting that the effect of metformin on C. elegans was mediated through an effect on E. coli protein translation. Using UV-killed E. coli, or acute treatments of the worms in the absence of drug are good ways to control for this possibility.
good luck,
Jordan
I just wanted to echo Jordan Ward’s last point: especially if you already suspect your drug has antimicrobial properties, you’re going to need to deliver it without affecting the bacteria. Exposing the worms to drug only in the absence of bacteria, growing worms axenically, and growing worms on killed E. coli are all options; the dead-bacteria option seems the most promising.
Hey,
Thanks for the advice, I think using the UV killed bacteria to grow the worms on will be the best option for me.
Do you by any chance have any references or protocols on using the UV killed bacteria or how to kill/store them? If so, please let me know where I can find them or email me at elisabeth.chang@kcl.ac.uk.
Best regards,
e.chang
Although there are probably a lot of protocols out there, I came across this one from Matt Kaeberlein’s lab in J Vis Exp.
http://www.jove.com/video/1152/measuring-caenorhabditis-elegans-life-span-on-solid-media?ID=1152
If you scroll down below the video there is a written protocol on how they make plates, including how they UV-kill the E. coli. You’ll need access to a UV Stratalinker 2400, or some similar piece of equipment.