I plan to use mouse mammary tumor virus-long terminal repeats (MMTV-LTR, it is a well-characterized mammalian promoter which can be induced by Dexamethasone) to express EGFP in MCF-7 breast cancer cells.
I bought pGL4.36 plasmid (it has a luciferase gene drived by MMTV-LTR) from Promega and substitute the luciferase gene with an EGFP gene. I used the new plasmid to transfect MCF-7 cells and induce with Dex for 6 hours.
When I observe the cells under a microscope, no green fluorescent signals were detected. I quantified the mean fluorescent signals in each cell and they are almost same as my control cells (transfected with the plasmid backbone), meaning that there might be not a single copy of EGFP being expressed.
The whole experiment seems straight-forward and I really don't know why it doesn't work. I'm wondering what can be possible reasons for this failure and what I can do for trouble-shooting.
Thank you very much!
I agree with the earlier reply. As Maureen J Ostaff said, first you need to know whether your transfection protocol is working or not (check with the control). Second, standardize your protocol using different transfection reagents (eg. lipofectamine, endofectamine, TurboFectin, etc.,) and find out which one is working.
Time for the transfection is also important, since I'm not aware of your procedure, if transfection is not observed may be you can further keep up to 2 hrs.
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