I want to use the pLL3.7 lentiviral vector (U6 promoter) to overexpress miRNAs. However, I have some doubts regarding the sequence that I have to clone in the vector to achieve overexpression. I think I have 3 options (please let me know if I said something wrong):
1) Clone the sequence of the pre-miR, bypassing the processing by Drosha. This would be equivalent to overexpress a shRNA, as it would already have the 3´ overhang necessary for nucleous export.
2) Clone the sequence of pri-miR. This would form a longer hairpin that has to be cleaved by Drosha to generate the pre-miR.
3) Clone the sequence of pri-miR + flanking sequences (+/- 200pb in both sides). This would be the longest sequence (~500pb), but would have a more physiological processing/expression (?). (Would U6 handle this long transcript??)
Assuming that I want to validate a new target, which of these would be the best option?
Thanks very much!
Hi Leonardo - Great question. I think you're best bet is to go with option #2 or, better yet, #3.
The issue here is that you need to make sure you get the exact pre-miRNA with proper folding so that Dicer cleavage with occur in the correct spot. This is a big concern with miRNA over-expression as a few base change in the cleavage site will shift which bases are in the "Seed sequence" region and, theoretically, drastically change the targets (see this paper: www.journallab.org/papers/8609) This is in contrast to siRNA which function using much higher complementarity, and which can have much more leeway in where exactly Dicer cleaves takes place while still retaining specificity in targeting ( check out, this reference for example - siRNA can have as few as 16bases complementary: www.journallab.org/papers/8608).
So, I think the real concern is, how will you know if you Dicer cleavage is occurring properly and in what proportions? Stem-loop RT-PCR might work, but if there was a shift in the cleavage site, depending on the direction of the shift, you might still get a cross-reaction signal. Further, even if you trusted you assay to be specific only to the properly cleaved mature miRNA, you wouldn't know how much "shifted" miRNA you were over-expressing. You could deep sequence, or clone out the small RNAs and sequence a bunch of them individually, but this might not be worth the effort. It seems to me the safest way to ensure proper pre-miRNA and get Dicer cleavage at the correct location in good proportions, is to express a long enough of the pri-miRNA sequence such that Drosha cleavage occurs normally, and subsequently, Dicer cleavage should as well. I seem to recall a paper than demonstrated biochemically that 50bp on either side of the hairpin was enough for consistent proper Drosha cleavage, but I couldn't relocate the citation just now (you might be able to find it in this review: www.journallab.org/papers/8610). You can also take the sequence you want to over-express and throw it into a RNA folding algorithm to see if it's at least predicted to fold correctly.
Another option is to transfect in mature miRNA mimic. This gives extremely high levels of over-expression, but it is transient - lasting 2-6 days usually, depending on the turnover of the cells you are using.
Hope this helps.
I suggest you try all these three stretagies, it just a problem of cloneing, according my experience, shorter is better; but I don't find a rule. Of course, you need to validate the expression of your miRNA by e.g. stem-loop RT-PCR.
Hi Leonardo - Great question. I think you're best bet is to go with option #2 or, better yet, #3.
The issue here is that you need to make sure you get the exact pre-miRNA with proper folding so that Dicer cleavage with occur in the correct spot. This is a big concern with miRNA over-expression as a few base change in the cleavage site will shift which bases are in the "Seed sequence" region and, theoretically, drastically change the targets (see this paper: www.journallab.org/papers/8609) This is in contrast to siRNA which function using much higher complementarity, and which can have much more leeway in where exactly Dicer cleaves takes place while still retaining specificity in targeting ( check out, this reference for example - siRNA can have as few as 16bases complementary: www.journallab.org/papers/8608).
So, I think the real concern is, how will you know if you Dicer cleavage is occurring properly and in what proportions? Stem-loop RT-PCR might work, but if there was a shift in the cleavage site, depending on the direction of the shift, you might still get a cross-reaction signal. Further, even if you trusted you assay to be specific only to the properly cleaved mature miRNA, you wouldn't know how much "shifted" miRNA you were over-expressing. You could deep sequence, or clone out the small RNAs and sequence a bunch of them individually, but this might not be worth the effort. It seems to me the safest way to ensure proper pre-miRNA and get Dicer cleavage at the correct location in good proportions, is to express a long enough of the pri-miRNA sequence such that Drosha cleavage occurs normally, and subsequently, Dicer cleavage should as well. I seem to recall a paper than demonstrated biochemically that 50bp on either side of the hairpin was enough for consistent proper Drosha cleavage, but I couldn't relocate the citation just now (you might be able to find it in this review: www.journallab.org/papers/8610). You can also take the sequence you want to over-express and throw it into a RNA folding algorithm to see if it's at least predicted to fold correctly.
Another option is to transfect in mature miRNA mimic. This gives extremely high levels of over-expression, but it is transient - lasting 2-6 days usually, depending on the turnover of the cells you are using.
Hope this helps.
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