co-transfection - co-transfection affect gene expression? (Jun/23/2006 )
Hi,
I have a problem with co-transfection of different genes in Hela cell line. Some case both genes express very well. In other case one gene express well and other gene suppress. When i did alone that suppress gene expresed very well in Hela cell line. So I have a question that co-transfection suppress that particular gene?
If any one have answer please let me know.
Thanks
KUMAR
I had the same problem when overexpressing E2F-1. It could really downregulate the expression of almost all the genes I cotransfected with it and all of them were under the control of the CMV promoter. I got crazy trying to normalize somehow (I did beta-gal assays and load according to the results). Finally I gave up and I'm always transfecting less E2F-1 than the other plasmid(s). In this way I don't see this phenomenon anymore.
Hope this can help.
I have a problem with co-transfection of different genes in Hela cell line. Some case both genes express very well. In other case one gene express well and other gene suppress. When i did alone that suppress gene expresed very well in Hela cell line. So I have a question that co-transfection suppress that particular gene?
If any one have answer please let me know.
Thanks
KUMAR
i think it may depend on the plasmids you are cotransfecting. if both drive expression from same promoters (like CMV) you can see competition. thats why i try to use different plasmids for contransfection, and always include proper empty vectors controls to see what is going on.
In my case it was a squelching phenomenon. One has to be very careful when working with transcription factors...
Hi,
Thank you very much for yours reply.
Yes, both of my plasmid vectors having CMV promotor site. So, as per your suggesstions i need to normalize my gene concentration. Can anyone help me to send the protocol.
Thanks
KUMAR