I'm trying to express a GFP-tagged transcription factor in H9C2 cells (rat myoblast). I've tried lipofectamine, superfect and electroporation and then checked for fluorescence but I see nothing. The plasmid I have code for the human protein, do you think that could be the problem? Thanks
Hi Mariana,
the codon usage would only affect the processivity of translation but not necessarily the protein product itself since both model and transgene are of mammalian origin. did you check the expression in HeLa too? I would definitively also check your transfected cells for the GFP tagged TF on western blot. sometimes GFP becomes mis-folded forms aggregates and thereby is inactivated, particularly if it is expressed too strong in a transfected cell, yet on western you would still be able to detect it.
Hope I could help
Hi Mariana, there may be several not necessarely trivial issues you might want to clarify:
- I assume that the same construct can be successfully transfected in other control cell lines. Is that so?
- Have you tried transfecting the backbone-GFP alone?
- Have you run a protein gel and western blot probing GFP and your protein of interest? Degradation may be an issue.
In addition I would suggest trying PEI transfection. There is some evidence it performs better than electroporation on rat cells.
I attached a link; I hope it works and wish you good luck.
http://www.ncbi.nlm.nih.gov/pubmed/21360090
Thanks for the answer! I have use this construct successfully before on RKO cells (human colon cancer). Beside not getting fluorescence signal another important difference with my previous experiment is now the transfection is very toxic, no matter which method I use. I'll check protein expression by western blot and will try transfect a different protein just in case mine is toxic. Thanks again!
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