I am generating a construct to make an animal line. I would like to express a marker before a mir30-based shRNA (CMV promoter - marker - mir30shRNA-SV40pA). How can I link this two elements so that the fluorescent protein is produced correctly and also does the shRNA?
Hi Anna, It´s neccesary to expressed de fluorescent protein and the shRNA from the same mRNA?. If that so, I think that construct will be completely or partially silenced, depending on the the saturation of the RNA silencing machinery. I think that you should express the Fluorescent protein from one promoter and the shRNA from another.
http://www.biotechniques.com/multimedia/archive/00036/BTN_A_04361ST02_O_36154a.pdf
http://www.sabiosciences.com/manuals/shRNA_brochure.pdf
Good Luck!!!
Hi Anna
Like Gabriel said it. They will both have to have their own promoter. You can replace your selection marker to express a fluorescent protein if that's what you want. That way you don't have to buy a new vector. If interested, I can send you a file with that cloning strategy if you want.
Good luck
Hi Anna
As you have suggested in your original message, you can express a fluorescent protein from the same transcript as a miR30 shRNA. The shRNA should be put downstream of the fluorescent protein, effectively in the 3' UTR of the transcript. This yields very good fluorescence and directly links it to shRNA production (and hopefully target gene knockdown). You can just clone the miR30 shRNA cassette downstream of the stop codon of the fluorescent protein. The miR30 cassette comprises approximately 120 bp either side of the XhoI and EcoRI sites that flank the shRNA insert, so the whole thing is approximately 350 bp with insert. So your suggested configuration should work.
This approach has been used extensively before but one of the best examples is attached.
Don't put the shRNA upstream of the fluorescent protein - anecdotally this seems to disable both.
Hope this helps.
Regards, Ross
Anna, If you want to have both the protein and the shRNA you need to have someway to stabilize the coding transcript once the shRNA is processed. You can do it by adding a stabilizing element or having your shRNA in an intron similar to the way done in the linked paper for gRNA.
Article Multiplexed and Programmable Regulation of Gene Networks wit...
Hi Anna
Like Yoav said, it is very easy to do that as an intron within the coding sequence of the reporter gene, as we did in our publication. The reporter protein will be translated only if there is proper splicing, which indicates that the miRNA is produced. Nevertheless, I recommend validating the miRNA production with a second construct that encodes an orthogonal reporter with your miRNA binding sites encoded in the 3'.
Also, the CMV promoter tend to be silenced relatively fast, so I would suggest using a native cellular promoter such as EF1a of UbC.
We will be happy to share our constructs with you.
best,
Lior
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