Hi All
Can anyone tell me if coinjecting a plasmid with a PCR fusion will give me a transgenic strain that expresses two of my proteins tagged to GFP and m-cherry ?I have heard that this is hard to get it to work.My other option is to make integrated transgenic strain for one and then inject the other. I would be glad to hear the feedback from people who actually tried doing it so that I can decide the best course of action.
Thanks in advance
and Merry Christmas!!
I would strongly advise against introducing one transgene into another transgenic strain, if you can avoid doing so; the results are somewhat unpredictable. I previously found that a rfp transgene that when injected into a non-transgenic strain gave good lines with strong RFP fluorescence gave lines easily but without RFP fluorescence when injected into a strain carrying a highly related gfp reporter. This sort of cosuppression had previously been reported in elegans and can cause something of a problem. Making sure the two transgenes are less closely relate to each other can help.
As to your first question, I’m a bit unclear: you have two reporter constructs, one a plasmid and one a PCR fusion, is that it? One drives expression of some protein fused to GFP and the other of some other protein fused to mCherry? What do you think would be the source of the problem? Normally this should work (though I find mCherry forms aggregates too easily). Maybe you’re concerned that one construct is linear and the other is circular? I don’t think this is a problem - if you can inject two separate plasmids, one plasmid and one PCR product should be fine (though, for other reasons, I’d always encourage you to clone your reporter and inject a known, homogenous construct).