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What is the best way to overexpress miRNAs?


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#1 leochicaybam

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Posted 17 October 2012 - 06:28 AM

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!

#2 pcrman

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Posted 01 November 2012 - 09:08 PM

The common practice is to clone the precursor miRNA (not pri-miRNA) with 100-200 bp flanking sequences.




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