shRNA to primary keratinocytes - (Feb/02/2012 )
I'm wondering how I can deliver shRNA into primary keratinocytes. I cannot use a growth medium containing lentivirus, because it contains serum and my cells differentiate when exposed to it. I've read about many kits that come ready (plasmid+transfecting agent, plasmid+virus), but the problem is that we have already ordered the plasmid kind of withoud wandering what to do next.
Does any chemical reagent that easily transfect the cells and doesn't kill them come to your mind? They have to be in a good condition because afterward the cells will be subjectet to quite an unpleasant treatment.
What do you think about applying lentivirus-containing medium onto the cells, centrufuging the plate and changing medium to keratinocyte growth medium after 1 hr (maybe the cells won't "notice" there is something wrong with the medium)
http://jcs.biologists.org/content/120/2/330.full <- they did this with retrovirus
concentrate the lentivirus and resuspend them in media without serum or simply PBS. This might solve your problem.