design parameters for multiple siRNA expression in vector - (Oct/29/2009 )

Hi everyone..
I wish to express siRNAs through vector.
my questions are
Is it possible to add sequences of more than one siRNA in tandem in a vector??
If yes then what should be the desired distance between the two selected siRNA to be diced properly by dicer??
Is their any specific feature of substrate (i.e.dsRNA) required for dicer to recognize n make a staggered cut to give mature siRNA or it acts on dsRNA without any particular recognition pattern..??


Why U6 promoter is always used in siRNA vectors... ??

ur replies will be of kind help as i am new in this field...

regards
nipun

nipun on Oct 28 2009, 11:59 PM said:

62 visitors yet no reply...
I wished to hear valuable suggestions from experts in the field...

Hopefully these are valuable suggestions to you...........

1. Since you have to use synthetic DNA oligos to generate shRNA clones, it may not be that easy to put several shRNA together just like miRNA cluster.
2. I am not sure a clustered shRNA, just like miRNA cluster, can be processed by Dicer without Drosha. If Drosha is required, then shRNA stem-loop is not sufficient for Drosha process.
3. It has been tested in mir-like shRNA cluster (e.g. 4 shRNAs in a row) expressed by pol II promoter, the first shRNA works the best....... so they don't work equally.
4. People including myself have used tandem repeats like H1-shRNA1-H1-shRNA2-H1-shRNA3-H1-shRNA4 to express multiple shRNAs in one construct.

The U6 and H1 are pol III promoters and they have been giving good shRNA knockdown results.

Dear Functional screens..
Thanks for ur really valuable suggestions...

some queries...

It means you are adding promoter in front of every shRNA..

I wish to express it in S pombe and S pombe doesnt have Drosha and till date miRNAs in S pombe are unproven so i am not very sure if shRNA will be processed in it or not..
I wish to design construct for a vector to transcribe siRNAs.
S pombe surely have a dicer and it helps in PTGS also {experimentally proven by Sigova et. al.}

So i wished to put potent siRNAs in tandem in the construct followed by its complementary sequence intervened by a loop sequence. So that it gives rise to a dsRNA which is processed by dicer to give siRNAs.
Is such a thing possible or not...?? so i wished to know Does dicer require any particular sequence to make staggered cuts in double stranded RNA...??

U6 used in mammalian cells is type III but Type III U6 promoter is not there in s pombe.
I am not sure about H1 promoters homologue in that...

Sorry, I am not a Dicer pro. My understanding is Dicer is a bit more like exonuclease III. You better check with literature.

I am not a S pombe. pro as well. my question is why not using long double-strand RNA which can be processed into small siRNAs by Dicer? Is there any particular reason you want to express shRNA?