I am currently trying to inject double stranded DNA as an alternative approach to do tissue specific knockdown. I am following a protocol in which DNA sense and antisense fused to tissue specific promoter are injected together with a marker. According to the protocol, I should inject the PCR product directly and at the concentration of 100 ng/ul. However, when I injected this mixture into the gonad, most of the worms don’t survive more than one day, which is not the case with the normal injection for generating the transgenic line. Does someone have any experience with this and can suggest me something that I should change or incorporate in my protocol to make the injection work?
If your PCR is already cleaned up, then it’s more likely to be #2.
There’s an easy control to test both possibilities: Try a similar construct (same promoter, different gene, such as GFP) before/after PCR cleanup via spin column. If neither is toxic, it’s probably your construct. If the first but not the second is toxic, it’s your PCR reaction.
Olaf Bossinger has had success getting this to work in the intestine by rescuing a rde-1 mutant (RNAi defective) with RDE-1 under the control of an intestinal specific promoter. You could do the same experiment but use your tissue specific promoter to rescue rde-1 instead. Then you can do RNAi with anything you want and get knock-down in a tissue specific manner.