Stable Transfection in Sf9 cells - Stable Transfection in Sf9 cells (Feb/10/2005 )

Hi i need help

i transfected a plasmid i made with the dna of interest. Pcr analysis shows that everything is okay with the plasmid.

i transfected sf9 cells with this plasmid trying to make stable cell line. after 72 hours post transfection, i checked for fluorescence using FACS and fluorescence microscopy. no detection at all.

Can anyone help.

thanks

If you want a stable cell line, try linearising your plasmid prior to transfection. This increases the rate of integration into the host genome.

If you think transfection efficiency is a problem, you could try NeoPhectin (http://www.expresson.com/productsandservices/neophectin.html) which works well in hard-to-transfect cell types.

thanks

you cannot stably transfect sf9 cells with bacmid because upon expression of viral proteins sf9 host cells go into scenesence and stop dividing. thus, you cannot have a stably transfected sf9 cell line that contains baculovirus unless you have a way of regulating expression of the viral proteins.

well for protein expression, i don't have good success with transfection. That's why i collect the supernatant of transfection for infecting other plate, collect the supernatant of it 3days after, repeat it one. The subsequent stock serves as virus collection for infecting cells.
You should do a time-course experiment, but generally, proteins expressing very well 48 - 96h after infection.