about pre-miRNA cloned into lentivirus vectors - (Dec/03/2009 )

Hello, everyone

I am trying to clone the premiRNA into lentivirus vectors, then to express the mature miRNA. How many flanking sequence of the 70 stem-loop (pre miRNA) need to be cloned into the vector at the same time? I read some papers, some advises that 200-300 bps upstream and down stream of the pre miRNA should be cloned, then lead to the cloned fragment about 500bp. In other papers, only about 100 bps upstream and downstream of the stemloop should be cloned, then totally about 300 bps fragment?

Which one is better for the further expression of mature miRNAs? Anyone has experience?
Thanks a lot in advance

veers

veer2 on Dec 2 2009, 11:25 PM said:

So what you're asking is how much you need of the PRImiRNA sequence? I recommend using this article as a basis for your system. I did, it worked fine

Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ. A lentivi-ral microRNA-based system for single-copy polymerase II- regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA 2005; 102: 13212-7.

One word of advice though; if you plan on inserting your own hairpin (which I guess is the case), be very careful if you include cloning sites at the ends for easy hairpin exchange as they can alter the secondary structure of the pre-miRNA. Use RNAfold at http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi to check that your structure folds up as the wild type.
Good luck!

Really thanks a lot for your quick and kind reply

I just use the 70 premiRNA sequence from Sanger base and want to add the flanking sequence of upstream and down stream of the pre miRNA. But usually how many flanking sequence of this stem loop should be included? 100bps or 200-300bps? any difference on the final mature miRNA expression? Do you have above experience?

Thanks a lot

veer2 on Dec 3 2009, 01:47 AM said:

Yes I'd also like to know what we should set the bps to in this situation. I'm guessing 200bps.

interesting problem important to me; tell me how it goes