I engineered an eukaryotic promoter inserting the LacO sequence. The system I designed renders the expression LacI dependent……my problem is that the expression is effectively silenced by LacI, but I failed in rescuing the expression by adding IPTG in cell media. Do you know if eukaryotic cells are able to process IPTG? In that case, what IPTG concentration should I use? I tried different concentration I found in literature, but no rescue was observed.
Thanks
If I understand the situation correctly, You assume that the Laci (repressor is being expression thus turning off the Lac O. Do you know if IPTG is penetrating the cells? In bacteria there is a permease transporter protein that allows for lactose (IPTG) to enter.
Of course, I am actually assuming that LacI is expressed (I copied the modified construct from literature, and thus including a nuclear localization segnal). The system seems to work fine, because the LacI is silencing my gene, but induction with IPTG is ineffective. Even treatment with galactose sugar is uneffective. Actually I don't know how to evaluate if IPTG is enetering into cells. Do you know how to evaluate this aspect?
Is there anything in the literature about lactose permeases in eukaryotic systems? It is probably fairly involved to assay for permeability.
Do you see expression from your LacO containing promoter when LacI is not expressed in the same cells?
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