What is the best way to overexpress miRNAs? - (Oct/17/2012 )
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!
The common practice is to clone the precursor miRNA (not pri-miRNA) with 100-200 bp flanking sequences.