I just had this question from my research associate:
would integration of an extrachromosomal array not expressing in the germline, but present in somatic tissues, potentially allow it to be expressed in the germline once it is integrated?
What do folks think? My gut feeling is that it would stay silenced, but if you could get an animal where the integrated array becomes expressed (maybe growing the animals at higher temp) then it could become stably expressed in the germline? I suppose it would also depend on where it integrates. My gut feeling would be to not spend the effort to integrate a transgene that doesn’t express in the germline in the hopes that integrating it gets around silencing of the array.
I don’t think integrating an array that’s silenced in the germline will cause it to become desilenced in the germline. I can’t even see what the mechanism would be.
If you want expression in the germline, you need to plan for it from the start: low-copy arrays or integrants (via gene gun), complex arrays, or single-copy insertions. The last is I think the most popular these days.
I believe you can desilence repetitive transgenes in the germline by interfering with nuclear RNAi (for example, transiently by RNAi targeting mut-7) but I’ve never done it and don’t know how well it works.
A high-copy, repetitive extrachromosomal array that’s silenced in the germline will still be silenced when integrated as a high-copy, repetitive array. The silencing is likely piRNA based, and lead to epigenetic silencing. Those epigenetic marks should still be present when the array is stitched into a chromosome following dsDNA breaks. I agree with Hillel, it’s best to start from scratch. You need to think about the 5’ end, the 3’ end and the introns. At minimum use a germline licensed promoter and utr (for example Pmex-5 and tbb-2, respectively), and do a single-copy insertion using mosSCI or miniMos.
Good luck!
Steve
Way back we integrated many arrays that should be expressed in soma and germline, but were only expressed in the soma.
Integration never produced germline desilencing. Even using bombardment and the pie-1 promoter, fewer than half of the
integrated lines give germline expression. Germline expression seemed to correlate with very low copy number. Single copy
transgene techniques are definitely the way to go.