Leakage of ER(T2)-fusion protein ? - in cell culture (Jun/16/2008 )
Hi,
I was wondering if somebody could give me some advice please...
I have "my" protein, fused to a mutated version of the estrogen receptor ligand binding domain (ER(T2).
(Due to the point mutations, the ER(T2) is insensitive to Estrogen, but highly sensitive to Tamoxifen. so basically in the presence of tamoxifen, the ERT2-cooupled protein can translocate into the nucleus and do its thing)
I tested the construct in P19 cells (cultured with serum in the media), and NS5, a neural stem cell line in defined medium containing progesterone/N2 supplement (which has a couple of other factors) and EGF/FGF. It's working in the sense that the construct is inducible and I get a really good activation level.
But it also seems to be a bit leaky (meaning I see activation in the absence of tamoxifen).
Does anybody have some experience with ER(T2)-fusions and can comment on the leakage-problem?
(I am actually wondering wether the leakage is inherent to the ER(T2) or the cell culture conditions or wether it's a problem of protein level)
Thanks!!
dedee
I was wondering if somebody could give me some advice please...
I have "my" protein, fused to a mutated version of the estrogen receptor ligand binding domain (ER(T2).
(Due to the point mutations, the ER(T2) is insensitive to Estrogen, but highly sensitive to Tamoxifen. so basically in the presence of tamoxifen, the ERT2-cooupled protein can translocate into the nucleus and do its thing)
I tested the construct in P19 cells (cultured with serum in the media), and NS5, a neural stem cell line in defined medium containing progesterone/N2 supplement (which has a couple of other factors) and EGF/FGF. It's working in the sense that the construct is inducible and I get a really good activation level.
But it also seems to be a bit leaky (meaning I see activation in the absence of tamoxifen).
Does anybody have some experience with ER(T2)-fusions and can comment on the leakage-problem?
(I am actually wondering wether the leakage is inherent to the ER(T2) or the cell culture conditions or wether it's a problem of protein level)
Thanks!!
dedee
did you check if your mutated estrogen receptor exhibits any binding activity to possible estrogens (estriol, estradiol, estrone)? even if there is any binding activity left, it may explain low levels of expression/activity;
moreover, some proteins are exhibited at a basic, non-induced, constitutive expression level
It's rendered inactive to estrogen(s) by mutagenesis...I don't really know how it will react to other substances (pregesterone/egf?)...
In this case leaking means that it is located in the nucleus, as ligand binding to the ER(T2) enables nuclear translocation. the protein is always expressed. I was wondering wether somebody here has any experience with that system...
Please?