Materials

For stable transfection:

• ice cold, sterile, serum-free HBSS or PBS

• expression vector containing gene of interest (10-30 mg)

• mammalian cell line (10 x 10⁶ cells; >90 % viable)

• sterile electroporation chambers

For transient transfections:

• sterile, serum-free HBSS or PBS at room tempersture

• expression vector containing gene of interest (20 mg)

• mammalian cell line (20 x 10⁶ cells; >90 % viable)

• sterile electroporation chambers

Methods

1. For stable transfection, wash cells in ice cold, serum-free HBSS or PBS twice and centrifuge at 250 x g (1000 rpm) for 5 minutes at 4^o C. If you are using adherent cells, trypsinize them according to standard procedures and add medium with serum to inactivate trypsin. Centrifuge the cells, aspirate the medium, and wash cells in serum-free HBSS or PBS as mentioned above. For transient transfection, follow the same protocol but perform all steps at room temperature.
   
    
2. Suspend the cells in 1 ml of serum-free HBSS or PBS and add the appropriate amount of DNA. The total volume of DNA should not exceed 10% of the total volume or 100 ml in order to maintain the correct osmolarity.
    
3. Transfer cells into an electroporation chamber and incubate cells and DNA for 10 minutes on ice for stable transfections and at room temperature for transient transfections.
    
4. Place the chamber in an electroporation apparatus and shock the cells at the desired voltage and capacitance settings. These settings will vary depending on both the cell line and the electroporation apparatus used. (In our laboratory, we usually use the following settings on a BRL Cell-Porator: 400 V and 60 mF for stable transfections and 350 V and 850 mF for transient transfections.) Let the cells recover in another 10 minute incubation.
    
5. Place the cells in a T25 flask containing 10 ml of prewarmed medium with serum, and place in 37^o C incubator.
    
6. For stable transfections, allow cells to grow in T25 flask for 24 hours. Then plate them at a density of 5-10 x 10⁵ cells/0.5 ml in 24-well plates, and allow them to grow for another 24 hours. It is important for the cells to grow in the absence of selection in order for them to recover from the electroporation procedure and to express the gene required for selection. On the next day, the selection reagent can be added. Add 0.5 ml of this reagent, at twice its optimal concentration, to obtain a final 1X concentration. Since every cell line is different, it is necessary to determine the optimum concentration of the selection reagent for each cell line (see next section). Feed the transfectants every 3 to 4 days with fresh medium containing the selection reagent. Positive transfectants, those that survive selection, can usually be visualized 6 to 14 days post transfection.

For transient transfections, cells can be maintained in the T25 flask and are ready for analysis 24 to 48 hours post transfection.

Determining The Optimum Selection Concentration For Stable Transfections

It is necessary to identify the optimal concentration of the selection reagent when performing stable transfections. The goal is to find a concentration for which it takes 3 days for all of the cells to die.

Methods

For ease of explanation, a sample titering scheme for the selection reagent G418 (to be used with vectors containing the neomycin resistance gene) follows.

1. Dilute cells (which have NOT been transfected) to 5 x 10⁵ cells/ml and plate 1 ml/well in a 24-well plate. Add G418 to a final concentration of 0.5 to 1.3 mg/ml.  Remember to keep one well to which no G418 is added.
    
2. Observe the cells and note their condition over the course of a week. The selection concentration that results in complete cell death 3 days after the start of the culture is the optimal concentration for generating stable transfectants using this cell line

©1995-2009 Loyola University Chicago Stritch School of Medicine.
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