Transfection of keratinocytes with siRNA - (Jul/12/2005 )

Hello,

I want to transfect primary kerationcytes with siRNA. Does anybody have experience on that? I grow the cells in serum-free KGM (Cambrex) and in the first try I forgot that one of the supplements is an antibiotic (Gentamycin). The cells died relatively quick after starting the transfection . So I tried it a second time with KGM without Gentamycin. The cells survived a little bit longer but died also . Now I read in some protocols that they even use a so called minimum medium which means only the basic medium containing no supplement s (like hydrocortisone, insulin) only EGF.
So, did anyone have already experience and success in transfecting keratinocytes with siRNA and if so, which transfection reagent and what conditions did you use?
Thanks in advance

Carsten

We have success in transfecting primary human keratinocytes using GenCarrier-1 reagent (epoch biolabs) on regular plasmid DNA (~45%) and siRNA (~65%). Cells were maintained in serum-free keratinocyte growth medium (KGM; GIBCO/Invitrogen) and were transfected ~ 4–6 weeks after initial preparation from foreskins at 70% density. We avoid serum, PBS, antibiotics during transfection and got very consistent results. In contrast to lipofectene or lipofectamine2000, gencarrier-1 displays very low toxicity if any. It is also the cheapest on the market for keratinocyte Tfx. good luck.

Thank you very much. I tried the protocol with lipofectamine 2000 avoiding PBS, serum and antibiotics (in fact everything but EGF )) and it seems to work, at least one time until now ;o).