Kozak Sequences and Mammalian Expression Vectors - (Oct/20/2005 )

So I've cloned a receptor gene into a GFP mammalian expression vector. But my problem is that the gene is toxic when highly overexpressed in cells. I haved transfected 2 different cell lines and it kills the cells every time.

I'm wondering if anyone knows of any tricks to lower expression. I don't what to use a Tet on/off system. I've never heard anything good about those systems anyway. And I worry about using an unstable GFP vector because I'm trying to study the trafficking of a receptor and do not want an unstable protein giving me a false degradation pathway.

Does anyone know of a way to lower expression by munipulating the kozak sequence or something similiar? I'm thinking of just leaving the kozak off, which may or maynot work. Know of any low expression vectors out there? Any other suggestions?

Regards

you can use core promoters for expression. I think of CMV core, or U1 core promoters that shows limited activity...

Dear Fattire,

you are going to have to go down the inducible promoter path.

Tet on-off has a few problems but it isn't the only system out there.

What cells are you using?