Use of dual promoter for siRNA - (Oct/01/2005 )

Hi,
has anyone used the dual promoter for designing the siRNA vector to be used in mammals?
please could you reply soon???
thanks
dinesh

hi
several vectors have benn designed and two dual promoters constructions were tested with various efficiency. First, each strand of the siRNA molecule was transcribed by a specific promoter.
In second one, the same sequence ws transcribed on both strand by two facing promoters. i can't remember at time if they were tested in mammalian organism, but they were successfully aplllied in mammalian cells.
But it's more eficient to transcribe an hairpin structure.
fred

this article may help you :
http://www.ncbi.nlm.nih.gov/entrez/query.f...4177&query_hl=3

Yeap the silencing efficiency of dual promoter system sometimes is not higher than hairpin construct. However, it is enough efficient for further studies, especially in genome-wide screens. The best advantage of dual promoter system is that it only require one step cloning without the mention of orientation. Therefore, you can clone cDNA libraries easily and it also save the time, labor for constructing the hairpin.

There is a cool alternative. A terminator was placed in a vector first in the sense direction then immediately in the antisense direction. This causes a short hairpin in the terminator (this was a Nos terminator). The cell machinery will then extend the RNAhairpin reagion tto the rest of the gene. Make your own mammailian vector like this for cloning and RNAi and kiss designing future hairpins goodbye!

<Gene of interest>------Nos-soN This yields (after transcription)

Nos-)
<Gene of interest>-------Nos-)

This is then extended in the cell to:

<Gene of interest>-------Nos-)
<Gene of interest>-------Nos-)

So your vector would need only

MCS--Nos-soN

And insert your gene and go!

Ooops, on the last post, the

Nos-)
<Gene of interest>-------Nos-)

Should have shown the two Nos on top of each other, having created a hairpin

Oh and the reversed Nos came first then the sense Nos. I guess otherwise it would terminate prior to the second Nos right, heh heh. My bad.

Hi,

I'm currently working with L4440, which is a vector for Andrew Fire's C. elegans RNAi kit. Basically, the MCS is flanked by converging T7 promoters. Thus, adding IPTG will induce transcription of sense and antisense strands of your insert which will anneal to yield dsRNA that corresponds to your insert.

This vector is intended for use in an RNase-III deficient strain of E. coli (HT115(DE3)). You can obtain HT115(DE3) from the C. elegans stock center in Minnesota and you can purchase L4440 from www.addgene.com for around $60.

Hope that helps,
Hank