How to overexpress miRNA? - (Sep/08/2006 )

Hi everyone!

Did you try any constructs to overexpress miRNA?Is there any differences from protein expression vector?

plus , how do you choose the miRNA context? I mean how many up and down stream of mature miRNA to be cloned into the vector is recomented?

THank you for your advices!

-zany-

one would use polymerase 2 promoter for protein expression.

For miRNA, in nature, they can be expressed by both polymerase 2 and polymerase 3 promoters.

If u want to express it by cloning into a vector, using polymearse 3 should b sufficient.

Polymerase 3 promoters - human H1, U6 etc

U need to select the miRNA from database or test out a couple.
I think cloning the exact miRNA is sufficient for expression.

-scolix-

http://www.nature.com/nmeth/journal/v3/n9/full/nmeth923.html
http://www.plantcell.org/cgi/content/full/18/5/1121

Sun D, Melegari M, Sridhar S, Rogler CE, Zhu L. Related Articles, Links
Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown.
Biotechniques. 2006 Jul;41(1):59-63.

-rye-

Do you want to express it in mammalian cells? If yes, the following link might help.
https://catalog.invitrogen.com/index.cfm?fu...0America%20Main

You can clone the dsDNA fragment (coded for the miRNA) into the vector, and then transfect into the cells.

If you would like to express it into other cell type, you might need to change the promoter.

-sallylyc-

Thank All of you very much!

I'm using Class II promoter ,and it seem to overexpress in my cell. I cloned 200bp context into the vector, and it worked!

-zany-

Hey,

I am also working on setting up over expression systems for miRNAs.
I have attached a good paper for you but just in case it doesnt open here's the name:

Sequence requirements for microRNA processng and function in human cells.
Yan Zeng and Bryan R.Cullen
RNA (2003) 9: 112-123

This is more or less the method I am using. These CMV promoted plasmids can be gotten from groups already working on this sort of thing or else you could probably already have it. CMV is a strong promoter helping you to overexpress

Hope this is of help

Any qs email

-cassiha-

Thank you very much Ihave already overexpressed my miRNA,Though a mild change happend after constrcut was delivered into host cell. May be it is becouse the efficiency of infection is not enough

-zany-