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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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stable transfection in primary cells
Posted by: christine (IP Hidden, New member, 1)
Date: September 7, 2005 01:50PM
I recently transfected primary mouse cells with a puromycin-resistant construct designed to decrease expression of a certain protein. I have successfully selected stable transfectants with puromycin and the cells are growing well. The problem is that by ICC I see similar protein expression in control and transfected cells. Any ideas of what the cause of my problem could be?
Re: stable transfection in primary cells
Posted by: 3'-UTR (IP Hidden, Unregistered user, )
Date: September 8, 2005 04:42PM
(1) I think the problem is your sequence (either antisense or shRNA) designed to knockdown your target gene. It's better to choose 3-4 sequences to test their inhibitory effect in transient transfected cells first before stable transfection.
(2) Another thing is how many clones (transfectants) you pick up? more than 10? and how many did you run ICC? It's hard to think all of your clones turn out to be negative if you have >10 clones. (3) Have you done RT-PCR or Western beside ICC?
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