eat-2(ad1116) sterility at F2 after alkaline bleaching

Hello all.

I’m working with eat-2(ad1116) and I am having serious sterility issues.

When I bleach a plate of gravid adults, the F1 generations hatch synchronized - however, these are sterile. They lay dead eggs. The funny thing is that anything in the eat-2 background (other double mutants that I have made) also show the same sort of phenotype.

I have been using the same protocol for numerous years and I have never seen this occur. I’ve tried the following but ALL HAVE FAILED.

  1. New NGM plates.
  2. New additives in the NGM Plates
  3. Reduced bleaching time (From 3 mins to 1min)
  4. Old stocks of worms
  5. Different peptone
  6. Removal of streptomycin/nystatin from the media.
  7. A new eat-2(ad1116) from the CGC.

Any help is appreciated !!

Hi mehulmvora,

I’ve had a quick go with our eat-2(ad1116) stock we received from the CGC (I haven’t outcrossed what I received from them) and spot bleached them onto a plate that also contained food (outside where I put the drop of bleach).

I didn’t quantify the number of progeny per adult, but I did not have the impression that any of the animals that hatched from the bleached eggs showed sterility issues. There were plenty of F2 animals that hatched from the eggs laid by the F1 animals.

The issue might therefore not be the bleach itself but the starvation-induced arrest in your F1 animals (that you use to synchronize the population). You could thus try to just bleach your animals and then put the egg prep onto a seeded plate/into liquid containing food. This will reduce the stringency of your synchronization somewhat but might be a price worth paying in return for a second generation of animals.

Hope that helps,

Toby