Transfection of inducible ER-fusion construct. - Estrogen in fetal calf serum? (Mar/22/2006 )

Hi,

I have an ER-fusionprotein expression vector which I transfect to my cells. Unfortunatly all cells die before I add tamoxifen to the cells and it is not possible to generate stable clones.
So I believe that there is an activity of the fusionprotein without adding tamoxifen. Has anybody experience with those inducible ER-constructs?
Is it possible that my fetal calf serum contains enough estrogen to induce the protein?

Thanks for your help.

Hi,

I have an ER-fusionprotein expression vector which I transfect to my cells. Unfortunatly all cells die before I add tamoxifen to the cells and it is not possible to generate stable clones.
So I believe that there is an activity of the fusionprotein without adding tamoxifen. Has anybody experience with those inducible ER-constructs?
Is it possible that my fetal calf serum contains enough estrogen to induce the protein?

Thanks for your help.

I might be very wrong here but i am pretty sure that the ER used for this purpose has cirtain point mutations. Now i thought that those also make it responsive to tamoxifen but not estrogen. I'm sure somebody here can clarify that.

The ER system works by retaining the protein, tagged with the ER, in the endoplasmic reticulum via, i believe, HSP70. So unlike with a transcriptional inducible system, such as TET, the protein is pressent in the cell. It is often used for transcription factors. It is kept out of the nucleus until induction with Tamoxifen. Then it is no longer retained and can be translocated into the nucleus to do its action.
I supose this strategy is relevant to your protein also.?

Do you have protocol how do this? I never heard of this before...

Thanks


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u have to strip regular calf serum as it contains estrogenic compounds. I never use straight serum for my e2 experiments. All use DCC stripped serum.
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Hi, all:
I have been working on an ER fusion cell line for months. I got good induction with 4-HT ( I mean after adding 4-HT all protein move to nucleus) twice at the beginning, then never can repeat the result again.
I ordered new 4-HT from Sigma, still no help. It seems just no induction.
Any ideas? I am so desperate.

DCC Calf Serum is sold by all good Cellculture ompanies.....
Its very expensive.....but I dont know if its a good Idea to strip it by your own....