I am attempting to design a plasmid that expresses two shRNA constructs under the U6 promoter and a protein under CMV. This limits my vector choice away from many of the commercially multi-shRNA options available, so I have to do it old-school.
From the literature, the most straightforward way is to directly design two hairpins (sense, loop, antisense, terminator) and put a spacer of about 130 base pairs between them.
However, details about the spacer are very sparse. Does anyone have any experience with designing this or a good reference? I would think it would need some sort of insulator that doesn't match known genomic sequences -
Anyone have a reference or basic protocol they don't mind sharing?
Hi Levi,
What you are trying to do might be a bit too complicated. Your 3' shRNA is bound to be susceptible to extensive processing before it reaches RISC. And you will end up with quite a large vector.
Why not do co-transduction (preferred) or co-transfection? You will need two different selectable markers for the protein and shRNA vectors.
Hi Levi,
I agree with Dirk that this may get a bit complicated but I think with trial and error you will get it; given that your two shRNAs of course are efficient.
Here is what I have done which may not be the best way out there to do it. I had two efficient shRNAs that were each driven by their own hU6 promoter (hU6--shRNA--Terminator Ts). When the shRNAs were spaced by about 15bp (they both had their own hU6) the efficiency of the first shRNA (i.e. 5-->3') was reduced by 50% of its usually high efficiency knockdown while the second shRNA produced good protein inhibition. However, when the two U6-shRNA sequences were spaced by 115bp the efficiency of the 1st shRNA increased back to almost normal. I have not experimented with >115bp spacer but I would suggest that in this case (i.e. two different U6-shRNAs) the bigger spacer the better.
That being said, since you are using the same promoter I am not sure exactly of the most efficient spacer length. Also, you probably don't want to put Terminator sequences after the first shRNA.
I would try one or all of the following experimentations:
1. Space your shRNAs using the same promoter with130bp as well as a shorter spacer and see the outcome.
2. Use two different U6-shRNA constructs (U6--shRNA--Terminator Ts) in the same vector spaced by at least 130bp.
3. As Dirk mentioned you can always cotransfect or cotransduce your two shRNAs constructs. This may be the better way to go if you are transfecting cells like 293T cells which can be done easily with calcium-phosphate transfections. However if you are transducing cells I think that one viral vector containing both of your shRNAs is better so you know for sure that each selected cell contains “both” of your shRNAs and not one copy of one shRNA.
hope that helped
Mahmood
Dear Levi, Mahmood,
Mahmood presents an elegant alternative - did the cloning go well, Mahmood? I have had some difficulties with some shRNA sequences, and can imagine that cloning two in a row might be even worse?
If co-transduction remains an option: viruses can be nicely titered, so you can aim for infecting cells with a relative overdose of the most important shRNA, and then calculate how much cells will also containt the second shRNA.
Dear Dirk,
you are right it can be a pain cloning multiple shRNAs (with almost similar DNA sequences) in one vector. The hU6-shRNAs were originally each in a separate pLKO.1 lentiviral vector. I then PCRed out the shRNAs from pLKO.1 with different enzyme sites (i.e. sticky ends, so I had to have 4 single enzyme sites for the two shRNAs) and moved them in a different lentiviral vector that I had previously cloned in the 4 new enzyme sites (this can be done by making small DNA oligos that base pair with sticky ends which can then be ligated in the vector).
the 15bp spacer was easier since it was already present in the final vector. However for the extra 100bp spacer I had to reclone one of my U6-shRNAs in a puC19 vector, sequence, then rePCR the U6-shRNA with the enzyme sites making sure to start 100bp upstream of the hU6-shRNA sequence in the pUC19 vector. Of course, in this long process you always have to make sure you don't have duplicate enzyme sites. it's a relatively longer and tedious process but can be done.
Mahmood
Please read this one. You might find some answers: Nat Proto 2009;4(9):1338-4).
Good luck!
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