What if my expressed protein is toxic for the cells... - (May/31/2010 )
I've performed transfection experiments on neuroblastoma (SK-N-SH cells) with a GFP fusion protein and I thought the transfection wasn't working because I got very few fluorescent cells.
But I've observed that, althought the transfection efficiency is low, the few cells expressing GFP alone (the empty vector) look well and show a intense cytoplasmic fluorescence, but virtuallly any cell is expressing the fusion protein.
When I electroporated the cells, the pattern of expression is the same (OK for empty GFP, none for fusion protein).
An idea is that I'm trying to express a protein that is toxic for the cells (is a kinase domain), but:
a) The level of expression and transfection efficiency of this fusion protein is wonderful in HEK and COS cells (not so good for CHO, with results resembling those of neuroblastoma)
When I transfected mutated fusion protein (non-active) results were so awful as for the wild-type fusion protein.
c) When I treated SK-N-SH cells with retinoic acid (to differenciate these proliferating cells into neurons), results didn't changed.
What experiments should I perform to confirm that the fusion protein is toxic or what could be happening in the cells?
have you considered cloning your gene of interest into Tet-off and Tet-on gene expression system