I transiently transfected HEK293 cells with nano-glo dual-luciferase reporter assay system to create cells with constantly produced luciferase. Then, I treated cells with set of concentrations of G418 (from 100ug/ml to 400ug/ml). I found that the reading increases with concentration compared to untreated control cells instead of being the same.
Because if you are using G418 as your selection marker you're going to select for cells that express luciferase and they will multiply, making the signal brighter compared to a sample where no selection took place
In my case, G418 is not a selection marker. It is transient transfection with WT-Luc plasmid for a positive control. It's assumed that G418 does not affect the luciferase expression.
Never worked with this assay before, so just brainstorming here: G418 is an antibiotic, so it should be toxic to your cells.
Is the luciferase gene expression coupled to any other genes that might get expressed as a response to the toxic G418?
Also: are you sure that the nano-glo dual luciferase reporter you transfected your cells with doesn't contain a neomycin resistance gene?
Looking forward to hear what you discover!
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