Dear worm members,
I am trying to make a transgenic line that has germline expression. Unfortunately I can’t use MOSsci or bombardement, which are the preferred methods of getting germline I would say. I have tried injecting with a linearized fosmid, linearized Rol marker and added cut N2 DNA, but no luck so far. Does anyone have some good suggestion about how to solve this problem? I got 9 complex lines from various injections, but none of them give me germline expression.
Thanks for your help!
Germline expression can be challenging. A few questions:
- Using a fosmid suggests that you’re attempting to express the gene flanked by its endogenous 5’ and 3’ sequences; correct?
- Have you checked that the fosmid is intact (i.e., does not contain a deletion)?
- What is the evidence that the fosmid should be expressed in the germline?
Without knowing the details, here are a few suggestions:
- vary the ratio of fosmid and gDNA during injections; some genes work only at low concentrations.
- replace the 5’ and 3’ flanks with elements known to function in the germline (e.g., pie-1).
- generate Mosci or bombarded lines in a different strain, then cross them into your strain of interest.
Thanks for your help and I’ll try to clarify some things for you.
- Correct and even stronger, I have proof that the fosmid actually expresses in the soma when I use a simple array.
- Based on the results of the experiments with the simple array I am confident that it is intact
- The gene that is on the fosmid is synthetic sterile with another gene. The mutant worms are balanced and are throwing also sterile worms and the fosmid should rescue the sterility, but it is not doing that. Since it is expressing in the soma and the problem is in the sterility I can only assume it is the germline expression that is causing the problem.
The problem is that I really need to have it non-integrated, because I would like to do a forward synthetic lethal/sterile screen with it. So I really need my worms to segregate worms with and without the rescueing fosmid.
Hope this helps clarify matters a bit and if anyone has also a good suggestion, I would love to hear it. Varying the fosmid DNA concentration and the gDNA is definitely something to consider.