For the past few weeks I have been attempting to generate transgenic worms using MosSCI. However, most of F1 transgenic worms that I generate only have paralyzed offspring. I inject EG6699 worms with a mix containing 25 ng/ul CFJ150, 50 ng/ul CFJ601, and 2.5 ng/ul CFJ90. All of the F1 transgenic worms that I get clearly express the co-injection marker. Does anyone know how I can fix this issue?
Although I do not have experience with MosSCI, I do have experience with MiniMos (which has the random insertion site, but the same fundament as MosSCI).
Here are some thoughts:
- Have you tested or do you know if your plasmid of interest (with the gene you want to insert) is toxic for the worm?
- Have you tested other insertion sites? (maybe that insertion site is disturbing the worm’s genome in a weird way…)
- Are all of your plasmids correctly purified? Are you using RNAse in the mix? (when you do the minipreps)
Hope any of this helps a bit!
I will echo some of Vicky’s advice and add a bit to it.
If you don’t get any Unc rescue at all from injecting some derivative of pCFJ150 (with your transgene cloned in) then your transgene is probably toxic - or something is wrong with the backbone vector. I would suggest you try the following:
(1) Digest and sequence the cbr-unc-119 fragment in pCFJ150.
(2) Try to inject pCFJ150 with no insert and verify that you can get rescue.
(3) Lower the concentration of your transgene. 10 ng/ul is fine and you can also go substantially lower - you’ll still get Unc rescue and single-copy insert.
Best of luck with it - Christian