I've generated a Dox-inducible transgene overexpressing cell line after lentiviral transduction followed by single-cell cloning. The problem I am facing is that only 20-30% of the cells express transgene on the application of Dox despite being a pure population from a single cell. The concentration of Dox I've tested is 1-10ug/ml. The results remain the same at all concentrations. The media components are pen strep. L-glutamine, Beta-me, DMEM, FBS, and NEAA. Can any of this act as a dox inhibitor? OR another concern is that we are using FBS that is not tetracycline free because being always cultured in this FBS, is it possible for cells to shut down the expression of transgene if they experience stress from the continues expression of the gene as the cell line doesn't express this gene in WT conditions.
Any comments would help!
Thanks in advance!!!
Insertion is random. Have you tryied with other clones that in theory inserted in other places of the genome?
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