Toxic Transgenic GFP line and RNAi effects

OK, I’m looking for suggestions.

I have a transgenic line expressing a GFP fusion to a toxic protein (call it toxic transgene TT::GFP) under the control of the PIE-1 promoter in the germline that is killing the worms. I was fairly lucky to recover lines, and have been able to propagate the line using GFP RNAi. However, this causes problems when it comes time to study GFP fluorescence. I have found that it takes several generations before GFP expression returns. I’ve been waiting for about 5 since I have been operating on the assumption that toxicity correlates with levels of transgene expression, but an alternative to that is that it takes a few generations to see toxicity after return of transgene expression. Also, I would like to be sure that different animals have worn out the RNAi effect equally and have equal expression for phenotypic characterization. The simplest approach to make my experiments more reliable would be to inactivate the GFP RNAi effect and recover GFP expression as quickly as possible. I have considered these ideas:

  1. RNAi feed a gene that is non-essential to swamp out the GFP RNAi with another exogenous RNAi. If this works, I could also treat with RNAi against genes I’m interested and see how they affect my TT::GFP. Any suggested genes to try? I imagine that it would have to mount a robust RNAi effect in the germline for it to swamp out the RNAi effect - if this is actually possible.

  2. RNAi generational RNAi genes such as rde-1. However, I will again have variation of resuming TT::GFP expression based on the efficacy of rde-1(RNAi) and if this prevents animals from mounting RNAi responses, it will cause issues for studying TT::GFP when I RNAi other genes.

Anyone have thoughts on these ideas, or further suggestions?



We have developed a new technique to express transgenes in embryos that kill worms. See our draft at BioRxiv: