non viral transfection - efficiency? (Aug/25/2008 )
I'm new to this field and I was wondering how efficient some of these non viral vectors can be with different cell lines, also, do any of you have any experience using any non viral transfection methods in vivo?
B, T, NK, DC - what type of cells are thinking about using? A far as NK...most of the cell lines are horible. Some report sucess with Amaxa kit. B and T lines can be electroporated with varying degrees of efficiency. Don't expect more than 30-40%. In our lab, we can do things like overexpression or reporter assays, but shRNA or siRNA delivery to give sufficient knockdown has been a challenge. May be able to offer more help - if you give more specifics. If you come from a 293 or Hela lab...you may have some disapoints awaiting you
I am planning on using some primary fibroblasts, some nih 3t3 cells and maybe down the road some pc12 and DRGs. What is the maximum efficiency for transfecting some of these types of cells?
I've working with 3t3 , DRG, and pain-in-the -butt ShSy-5Y.... using lipofectamine (2000) I've gotten around 40-50% efficiency.
Depends on the gene being transfected, but there are a huge range of non-viral methods... lipid-based (lipofectamine range (2000, RNAiMax, LTX etc) and fugene are the common ones but there are plenty of others), calcium phosphate transfection, electroporation, gene-gun, all with different transfection efficiencies and good sides and bad sides.
It will also depend on the cells being transfected, some are easier than others. I routinely use SaOs-2, MCF7, Hela, 293, HCT116, K1, A549, SkMel series, 3T3, and T47D. All of them transfect OK, but some better than others. Playing around with the concentrations of DNA/RNA and transfection reagent helps a lot.
Incidentally, I have found that lipofectamine range, apart from RNAiMax all damage the cells a lot, but fugene doesn't.