apoptosis upon over-expression with pcDNA6-cDNA - how prove sequence specificit - (Jun/22/2011 )

Hello everyone,

I am trying to establish a cell line stably over-expressing my gene of interest. Transfecting the cDNA in a pcDNA6-plasmid lead to apoptosis of the cells with a 3 day delay compared to untransfected controls. Is there any good way to demonstrate clearly that this is sequence specific and definitely not related to transfection efficiency? Or a first step before I'd start cloning my cDNA into other (i.e. inducible) systems or similiar?

Any help would be really appreciated!!

Did you do an "empty" vector control (i.e. pcDNA6 with no insert) and a non-target vector control (i.e. pcDNA6 with another cDNA or a scrambled version of your cDNA)?

bob1 on Thu Jun 23 02:08:29 2011 said:

Hello,

Yes, I had the empty vector, which was proliferating happily under selection but no scrambled cDNA. How could I make a scrambled version of my cDNA?

Thanks so much for any help!

You can use another gene as a "non-target" control - if the cells die when you transfect this in, then it is a transfection issue, not a property of your gene.

You should also check expression of your gene in the cells by western and/or RT-PCR.

Thank you very much! I will measure mRNA expression and transfect another gene in parallel as a first step ).