I’ve been facing a problem with my transfection and I would really appreciate your help.
I am trying to transfect a mouse gene into CHO-K1 cells using lipofectamine 2000. My vectors are pcDNA 3.1 Hygro. As transfection efficiency control, I use pcDNA GFP. I transfected the cells in different ratios from 1:1, 1:2, 1:3, 2:2, 2:3. I usually transfected the cells in the evening and the day after I change the medium in order to avoid cytotoxicity and remove the dead cells. Between two or 3 day after transfection I split the cells and start the selection with Hygromicin B (500µg/ml).
The problem is when I test these cell in PCR they often are positive to my gene but when we try to detect the protein expression by FACS, this is very low (for CHO cells…around 20 or 30%). I don’t know what should I change or do in my transfection protocol.
I sequenced my full vector and it is ok. I sequenced the promoter region and also was ok. I tried Lipofectamine LTX and Roche Fugene HD that they claim to be less cytotoxic but I had the same results. I heard that the linearized vector the transfection would be worst but the genome integration is better. I tried this procedure and I had the worst results…
I don’t know anymore what should I change or what should do to get high percentage of transfected-expressing cells. This is not normal for CHO-K1 cell.
I would really appreciate your opinion