DEAE-dextran TRANSFECTION PROTOCOL

This is a generic DEAE-dextran transfection protocol optimized for GeneSwitch studies. It indicates our standard ratio of GeneSwitch to inducible transgene plasmid as 1:1. It also details the use of the ligand MFP to activate the system.

1. Plate HeLa/Cos-1 cells at 2 x 10⁵ cells/well on 6-well plates. Transfect cells ~24 hrs after plating.
2. Use 0.3 mg of the GeneSwitch plasmid with 0.3 mg of the inducible transgene plasmid per well.
3. Add DNA to 490 ml of PBS (Ca⁺⁺, Mg⁺⁺ free) containing 250 mg/ml DEAE-dextran. (DEAE-dextran stock solution is 10 mg/ml). If doing 3 wells/construct usually make enough for 4 wells. Therefore add the DNA to a 2 ml DEAE-dex/PBS soln.
4. Wash wells once with 2 mls of PBS (Ca⁺⁺, Mg⁺⁺ free) and add 500 ml of the DNA/DEAE-dextran/PBS mixture.
5. Incubate 30' at 37^oC, with rocking every 5'.
6. Add 2 mls of media (DMEM w/ 10%FBS, 1% P/S) containing 80 mM Chloroquine, incubate an additional 2.5 hrs at 37^oC. Make a Chloroquine stock soln at 10 mM in PBS.
7. Remove media and shock cells w/ 1 ml of 10% DMSO in media (DMEM w/ 10%FBS, 1% Pen/Strep) for 1'.
8. Remove DMSO solution and replace with 2 mls of media (DMEM w/10%FBS, 1% P/S). If treating cells let them incubate for 24 hrs before adding MFP at a concentration of 10^-8 M. (Usually replace the media in the well with media containing MFP at the proper concentration). Perform reporter assay 12-16 hrs after treatment.

Note: We have also found that the FuGene 6 transfection reagent from Roche Molecular Biochemicals, used as described by the manufacturer, works well with this system.