miRNA expression vector - (Dec/06/2008 )
Hi all, I’ve got a bit of a problem with my microRNA expression vector and maybe someone can give me a hand with this…
I’m trying to construct a mammalian expression vector that would overexpress my microRNA of interest in tissue culture cells (human).
So I’ve amplified pre-microRNA sequences (~300bp) that would contain the stem loop sequence of the desired miRNA from human genomic DNA. Inserted these sequences into pGEM-Easy, and subcloned the insert into pcDNA3.1(-). Verified the plasmids by sequencing – everything is in the right orientation with regards to the CMV promoter.
When I transiently transfect my plasmids into my cells (established protocol) and verify the expression of mature microRNA by qRT-PCR, I don’t see a difference from my control (untransfected cells or cells transfected with pcDNA3.1(-))!!!!!!!!!!!!!!!!!!!!!!
When I transfect, I include a co-transfection control which expresses renilla luciferase and I know that it is expressed.
Any suggestions? How can I make microRNA overexpression work???
I am also interesed with answer to your post.
I have the same problem.
I see the GFP from the construct and do not see the miRNA overexpresion.
Has anyone the same problem and find the solution to that?
We have also used pCDN3.1 vector to clone pre-miR and found exogenous miR expression is not very high ( a maximum 5-fold increase). Probably pCDNA is not the right vector for miR expression. Before you swith to another vector, make sure your transfection efficiency is OK. Your co-transfection luciferase vector may not tell how efficient your pCDNA vector has been transfected.
I used pSUPER.retro.puro from oligoengine. it works very well also in transfection, you don't need a very lon pre-mir insertion (it works VERY well also with 15-20 nt at each side) that enable you to skip the pcr step and to order a 130-140 oligo couple with sticky ends. you just anneal, clone, test by digestion and sequence.
very easy and strightforward