Cloning multiple miR30 based shRNAs into a Lenti - (Mar/06/2009 )

Hi there, has anyone tried cloning multiple miR30-based shRNAs into a Lentivirus?

I want to clone in a reporter (mCerulean/tdTomato) and three different shRNAs in a mir30 format.

1. can i use just the one promoter, or do i have to use two?
2. do i have to separate the miR30s by a stretch of sequence?
3. is this fundamentally flawed???

Any help would be great!

Thanks

If you want to express a miRNA, current consensus is to clone the precusor miRNA plus some flanking sequence because shRNA formatted miRNA may not fold and be processed properly.

Why do you want to clone multiple copy of the same miRNA? want to obtain high expression?

It looks like you are using polII promoter to express shRNA with mir-30 flanking sequences, am I right. If so, it should work just like expressing miRNA cluster. But you might pay attention on recombination due the repeat sequences.