Problems with plasmid transfection into primary keratinocytes - (Nov/09/2006 )

Hi!
I'm having problems with transfection of plasmid DNA into human primary keratinocytes. I have used Lipofectamine, but after 24h post-transfcetion all the cells were dead. I have fallowed the protocol from Invitrogen, which suggests changing the medium for complete medium after 5h. What can be the couse, maybe I have used too much DNA or too much Lipofectamine, which of those factors is more important. Thank you for your time and help

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usual the transfection reagent is toxic, try less. But the recommendations from invitrogen should work. Other possibility could be that your protein is pro-apoptotic ! Would be very interesting, try a time-course with different time points.

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perhaps LF is not the right choice; for our primary epithelial cells Qiagen transfection reagent was better than others; nevertheless, chemical transfection rates for primary epithelial cells are in general much lower than for cell lines, so lowering the concentration may not be useful; if chemical transfection will be a persistent problem, try classical CaCl2-method;

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As suggested by the previous guys, the modeification of transfection itself will be the first thing to do. However, I highly recommend to keep in your mind that the efficiency of transfection is very limited when you use the regular transfection to primary cells. If it is enough to detect the expression of target genes even though the limited population of cells, it would be all right. However, if you are planing to do bio-assay after thet I highly recommmend to move to alternatives such as retro- or adenovirus. Hopefully, this info will help your lab work.

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