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Linear DNA transfection - Daudi cells (Nov/18/2008 )

Dear mates,
I am trying to transfect daudi(B cell line with a 9 kb plasmid using amaxa nucleofection kit, and i am not succeeding at all.
Any body know any thing about transfecting the linear DNA instead???????????????? and how efficient it is???

Best regards

-thegen-

QUOTE (thegen @ Nov 18 2008, 08:19 AM)
Dear mates,
I am trying to transfect daudi(B cell line with a 9 kb plasmid using amaxa nucleofection kit, and i am not succeeding at all.
Any body know any thing about transfecting the linear DNA instead???????????????? and how efficient it is???

Best regards


Have you called Amaxa? They are usually ready to bend over backwards to get a transfection working. Nice to see good customer service . . .

They will probably suggest trying different program/solution combos with their GFP control vector.

As for transfecting linear dsDNA, it can be done with conventional reagents. I have only done it a couple times, with easier cell lines than B cells, but it can be done, although at lesser efficiency. The main concern is watching out for immune response, as linear dsDNA in the cytoplasm/endosome is a substrate for TLR3 and can induce IFN.

-jtotheizzoe-

QUOTE (jtotheizzoe @ Nov 18 2008, 08:24 AM)
QUOTE (thegen @ Nov 18 2008, 08:19 AM)
Dear mates,
I am trying to transfect daudi(B cell line with a 9 kb plasmid using amaxa nucleofection kit, and i am not succeeding at all.
Any body know any thing about transfecting the linear DNA instead???????????????? and how efficient it is???

Best regards


Have you called Amaxa? They are usually ready to bend over backwards to get a transfection working. Nice to see good customer service . . .

They will probably suggest trying different program/solution combos with their GFP control vector.

As for transfecting linear dsDNA, it can be done with conventional reagents. I have only done it a couple times, with easier cell lines than B cells, but it can be done, although at lesser efficiency. The main concern is watching out for immune response, as linear dsDNA in the cytoplasm/endosome is a substrate for TLR3 and can induce IFN.

Thanks for the answer biggrin.gif , i called amaxa and we are trying different things togather with them, but such optemization takes long time.....thats why i am going to linear DNA approach, Do you recommend a protocol or a transfecting reagent? and any things that you have to watch???? and how you can wach out for the immune response?????
thnks in advance

-thegen-

QUOTE (thegen @ Nov 19 2008, 10:37 AM)
QUOTE (jtotheizzoe @ Nov 18 2008, 08:24 AM)
QUOTE (thegen @ Nov 18 2008, 08:19 AM)
Dear mates,
I am trying to transfect daudi(B cell line with a 9 kb plasmid using amaxa nucleofection kit, and i am not succeeding at all.
Any body know any thing about transfecting the linear DNA instead???????????????? and how efficient it is???

Best regards


Have you called Amaxa? They are usually ready to bend over backwards to get a transfection working. Nice to see good customer service . . .

They will probably suggest trying different program/solution combos with their GFP control vector.

As for transfecting linear dsDNA, it can be done with conventional reagents. I have only done it a couple times, with easier cell lines than B cells, but it can be done, although at lesser efficiency. The main concern is watching out for immune response, as linear dsDNA in the cytoplasm/endosome is a substrate for TLR3 and can induce IFN.

Thanks for the answer biggrin.gif , i called amaxa and we are trying different things togather with them, but such optemization takes long time.....thats why i am going to linear DNA approach, Do you recommend a protocol or a transfecting reagent? and any things that you have to watch???? and how you can wach out for the immune response?????
thnks in advance


As for reagents, I always use Lipofectamine 2000. But that doesn't mean it is best for your cells. I deal with easy to transfect cells. I could essentially just wish the DNA in and it would go in. B cells are notoriously hard to transfect, though. I think DMRIE-C from Invitrogen does ok, and the new Lipofectamine LTX. Sorry I can't be more specific, there are just too many reagents on the market these days, and so many of them are cross-licensed that it is hard to keep track of what is what. It will just take a few phone calls to ask the companies about B cells.

To check the immune response: First, your cells will be dying en masse. But for a better answer, you can do a western for phosphorylated-Stat1, which is a downstream IFN target, or you can ELISA for interferons, or you can RT-PCR for some of the ISGs. There's lots of ways. Even kits. But remember, not all cells have a response and every cell type has a different pattern of immune gene expression.


-jtotheizzoe-