Method

1. Harvest exponentially growing cells (e.g., CHO cells) by trypsinization, and replate them at a density of 5 x 105 cells per 90-mm tissue culture dish in 10 ml of MEM-«, containing 10% fetal calf serum. Incubate the cultures for 18-20 hours at 37°C in a humidified incubator in an atmosphere of 5-7% CO2.

2. Replace the medium with 3 ml of warmed (37°C) medium containing serum, DNA (5 ng to 40 pg; no carrier DNA), and 30 pg of Polybrene. Mix the DNA with the medium before adding the 10 mg/ml Polybrene. Return the cells to the incubator for 6-16 hours. Gently rock the dishes every 90 minutes during the early stages of this incubation to ensure even exposure of the cells to the DNA-Polybrene mixture.

3. Remove the medium containing the DNA and Polybrene by aspiration. Add 5 ml of 30% DMSO in serum-containing medium. Gently swirl the DMSO medium around the dish to ensure even exposure of the cells to the solvent and place the dishes in the incubator.

4. After 4 minutes of incubation, remove the dishes from the incubator and immediately aspirate the DMSO solution. Wash the cells once or twice with warmed (37°C) serum-free medium, and add 10 ml of complete medium containing 10% fetal calf serum. If a sodium butyrate boost is to be included, then proceed to Step 5. If not, incubate the cultures for 48 hours at 37°C in a humidified incubator with an atmosphere of 5-7% CO2. Then proceed directly to either Step 6 (to assay for transient expression) or Step 7 (to establish stable transformants).

5. (Optional) To facilitate the transfection of cells treated with DMSO and Polybrene:

a. Add 500 mM sodium butyrate directly to the growth medium to a final concentration of 2.5-10 mM.

b. Incubate the cells for 20-24 hours at 37°C in a humidified incubator with an atmosphere of 5-7% CO2.

c. Remove the medium containing sodium butyrate, and replace it with butyrate-free medium containing 10% fetal bovine serum. Return the cells to the incubator.

6. If the objective is stable transformation of the cells, proceed directly to Step 7. For transient expression, examine the cells 1-2 days after transfection using one of the following assays:

• If a plasm id DNA expressing E. coli -galactosidase was used, follow the steps outlined in Chapter 17, Protocol 7, to measure enzyme activity in cell lysates. Alternatively, carry out a histochemical staining assay as detailed in the additional protocol in Chapter 16, Protocol 1.

• If a green fluorescence protein expression vector was used, examine the cells with a microscope under 450-490-nm illumination.

• For other gene products, analyze the newly synthesized protein by radioimmunoassay, by immunoblotting, by immunoprecipitation following in vivo metabolic labeling, or by assays of enzymatic activity in cell extracts.

7. To isolate stable transfectants: After the cells have incubated for 48 hours in nonselective medium (to allow expression of the transferred gene[s] to occur [Step 4]), either trypsinize or replate the cells in the appropriate selective medium or add the selective medium directly to the cells without further manipulation. Change this medium every 2-4 days for 2-3 weeks to remove the debris of dead cells and to allow colonies of resistant cells to grow.

8. Thereafter, clone individual colonies and propagate for the appropriate assay.

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