test fuGENE 6 for the large DNA - (May/07/2008 )

I am going to use FuGENE 6 for the first time on HEK293 cells.

Question 1, May I get a worked portocol from someone or somewhere?

Question 2, I am going to work on transfecting Adeasy system linearized 30-40kD DNA into HEK293 cells for packaging the virus. anyone knows can FuGENE6 work on larger DNA like that? any suggestion for that?

Thanks

I think this should work.

I used FuGene6 to transfect 293 cells with adenovirus plasmid (~ 33 kb) (BD systems) for packaging the virus. I used their recommended protocol (for 6cm dishes if I am not wrong, was a long time ago but I can look it up for you if you want). The transfection efficiency was not very high. I tried different ratios of DNA:Fugene but it did not improve much in my case (anyway, after the packaging you have to infect and reinfect the cells to get higher titration of the virus, right?). After transfection the cells were looking okay. I tried Lipofectamine2000 and calcium phosphate as well but somehow the cells looked like blown up and not very happy after transfection, so I continued with FuGene6.

Maybe it would be worth to try the transfection first with cells on coverslips. Then you could fix the cells at different time points after transfection and do immunofluorescence. That could give you an idea about the transfection efficiency and also if your construct is correctly expressed.

Thanks. I tried with regular plasmid ~5kb with FuGENE6 (according to manual). 3:1 gives me better transfection rate compare with other ratio. But it is still about 50% after 18hours to check GFP under microscope.

Do you mind let me know if you can package out the primary adenovirus with the "not very high transfection efficiency"?

Thanks

Oh, I worked with adenovirus in my old lab. So, I don`t have any constructs here.

Sorry that I couldn`t help you much.