4 replies to this topic

[veer2]

How to overexpress star miRNA (miRNA*) from pre-miRNA construct

I am working on miRNA* field. I plan to clone the pre-miRNA (about 500 bps with flank sequence) into vectors, then express mature miRNA in which the miRNA* is only by product. But, how to construct the pre miRNA vector in which the MAIN PRODUCT is miRNA*, not mature miRNA. I have no any clue on it and no papers on it.

Thanks in advance

[Functional Screens]

My two cents.............

My suggestion is using antisense inhibitor to block the dominant counter-mir from your pri-miRNA expression.  But by doing this, I still don't think the mir* is "OVEREXPRESSED" (if this is what you want?), the mir*just becomes more dominant.  The similar way is to change the C on the counter-mir to U (T) to eliminate its function but keep the base-pair.

You can also try to make siRNA-like double-stranded RNA with GC base pairing in one end to favor the miRNA machinery to take the mir* sequence, however, DICER might chop your "siRNA" into 19-mer and I think that's the drawback.  Alternatively, if you can change the base-pairing in the pre-miRNA to make mir* being favored, then it might work.

Comments..............?

[miRNA man]

You could also try a miRNA sponge approach to sequester mature miRNA but leave miRNA* unaffected.

[veer2]

Thanks a lot. To functional screening, in your post, you said maybe to change the basepair in pre miRNA to make the *miRNA as favorite product. What is the principle I should use to change the basepaire? Are there any papers to discuss about this problem?


Thanks again in advance

Edited by veer2, 25 October 2009 - 09:22 PM.

[Functional Screens]

veer2, on Oct 25 2009, 10:19 PM, said:

Thanks a lot. To functional screening, in your post, you said maybe to change the basepair in pre miRNA to make the *miRNA as favorite product. What is the principle I should use to change the basepaire? Are there any papers to discuss about this problem?


Thanks again in advance

Zamore rule.
Cell. 2003 115 199 Asymmetry in the assembly of the RNAi enzyme complex
Cell. 2003 115 209 Functional siRNAs and miRNAs exhibit strand bias