Can shRNA be used in one transcript with other gene - (Mar/28/2007 )
construct1: LTR-gene1-IRES- gene2
Gene 2 is the activator of a shRNA promoter.
construct2: U6 - shRNA
Then TAT activates construct 1, and gene 2 from construct 1 activates construct 2 and sh RNA is expressed.
Now, I need to solve problems with basal expression of U6 promoter and its activation.
this could also work. But verify the stability of these control elements for shRNA . they can be leaky.
I think here is the answer!
http://www.ncbi.nlm.nih.gov/entrez/query.f...p;dopt=Abstract
its a tet inducible one.
couldnt you have all of required elements in a single vector.
Sorry, i don't know what you mean.
LTR promoter is TAT inducible, this one is tet inducible.
edit: i did not read carefuly... this will not work, because i can't put a gene coding for tet in a cell. So, it can't be activated.
Is there a polIII promoter that is inducible by some protein that is not normally present in an animal cell.
There are only tet inducible promoters, but i need a protein inducible.
scolix:
thanks for all your help, but we didnt understood each other.
I was talking that i would like shRNA transcription in vivo from the host DNA and you were talking about vector based shRNA.
I am sorry because i was not clear enaugh.(and really confused)
And in this case you do not need a pol III promoter, but just a normal pol II.
thanks for all your help, but we didnt understood each other.
I was talking that i would like shRNA transcription in vivo from the host DNA and you were talking about vector based shRNA.
I am sorry because i was not clear enaugh.(and really confused)
And in this case you do not need a pol III promoter, but just a normal pol II.
I am confused because the article you referred was about a pol III promoter U6, which is tet regulatable.
Like I said, I was confused 
http://www.ingentaconnect.com/content/klu/...000001/00003965
It can work also with pol II promoter.
http://www.ingentaconnect.com/content/klu/...000001/00003965
It can work also with pol II promoter.
cool, thats interesting !!!
may b this solves your problem.
There are only tet inducible promoters, but i need a protein inducible.
Elledge and colleagues have developed a form of shRNA expression system that closely mimics the processing of endogenous miRNA--it's called shRNAmir. These casettes can be driven by a normal pol II promoter, and basically consist of your 19-mer siRNA (sense and antisense) strands separated by a miR-30 loop, flanked on both ends with the stem from miR-30
5' [125bp miR-30 stem]--*siRNA sense--[miR30-loop]--siRNA antisense*--[125bp miR-30 stem, antisense] 3'
(the things enclosed in asterisks are all that you actually clone in)
they're used in the vector pPRIME, among other "second generation" lentivirus vectors; I'd just get one of those and clone out the miR-30 regions and ligate them into your own vector.
References for this are Stegmeier F et al. PNAS. 102(37):13212-7 (2005).