Retroviral-Mediated Transduction: A Method to Introduce Target Gene into Cancer Cells

1. Transduction

1. Retroviral plasmid transfection
   1. Freshly thaw HEK293 cells the afternoon before in a water bath set to 37 °C. Spin the cryotube containing the HEK cells at 435 x g at room temperature for 3 min. Remove the supernatant via a vacuum and suspend the cell pellet in 1 mL of DMEM supplemented with 10% FBS, penicillin, streptomycin, and glutamine.
   2. Count the cells and add the appropriate volume to a 10 cm treated dish (~4 x 10⁶ HEK293T cells). Add 14 mL of the DMEM 10 media to the dish and mix it well by sliding up and down for an equal distribution. Place the dish in a 37 °C, 5% CO₂ incubator overnight.
   3. On the next morning, check the confluence of the cells under an inverted light microscope (50% or more works well).
   4. Combine the DNA (desired retroviral plasmid), PclEco (packaging plasmid), 2 M CaCl₂, and H₂0 in a 1.5 mL tube (= 500 µL) using a micropipette in the following amounts: 7.5 µg of DNA (BCR-ABL1-YFP), 7.5 µg of pCLeco, 62 µL of 2 M CaCl₂, and water to a final volume of 500 µL. Add 500 µL of 2x HBS to another 1.5 mL tube.
   5. Add the DNA mix dropwise to the 1.5 mL tube containing 500 µL of 2x HBS (280 mM NaCl, 100 mM HEPES, and 1.5 mM Na₂HPO₄) while mixing. Achieve this by setting a vortex to the lowest speed and hold the HBS tube on it for constant mixing while adding the transfection mix.
   6. Allow the transfection mix to incubate for 20–30 min at room temperature. During the incubation, add 25 nM chloroquine to the HEK cells and place them back in the incubator. After the incubation, add the transfection mix dropwise to the HEK cells and, then, gently swirl the media to mix the transfection mix throughout the plate.
   7. Incubate the cells with the mix for ~8 h in a 37 °C, 5% CO₂ incubator. Change the media on these cells by very slowly adding 14 mL of fresh DMEM 10 media. Pipetting slowly against the wall of the dish helps to prevent any stripping of the HEK cells. Incubate the cells overnight in the 37 °C, 5% CO₂ incubator.
   8. Collect the viral supernatant with a 10 mL syringe, draw the supernatant into the syringe, and attach the 0.45 µm syringe filter. Plunge the viral supernatant into a new collection tube. Take the first collection of viral supernatant ~16 h later.
2. Fibronectin viral concentration and transduction
   1. Add 2 mL of a 0.1 mg recombinant human fibronectin fragment in 1x PBS to untreated 6-well culture plates. Prepare as much fibronectin as needed, which depends on the number of cells and genotypes. One well can sufficiently transduce 10 million c-Kit+ cells. Incubate the plate overnight at 4 °C.
   2. The next day, remove the fibronectin from the wells with a pipette, pipette 2 mL of sterile 2% BSA in 1x PBS to block the plate, and incubate for 30 min at room temperature. Remove the BSA via a vacuum and wash thoroughly by pipetting 2 mL of 1x PBS then remove it via a vacuum.
   3. Immediately add freshly collected and filtered (at 0.45 µm) viral supernatant up to 5 mL. Centrifuge at 435 x g at 32 °C for 2 h. Remove the supernatant via a vacuum and repeat this step with additional freshly collected viral supernatant and spin again. Remove the supernatant via a vacuum and wash the wells by adding 2 mL of 1x PBS; then, remove it via a vacuum.
   4. Add the c-Kit+ mouse cells that were cultured overnight by spinning down the cells and resuspending the pellet in complete IMDM ,for culture. Culture these cells overnight in 2 x 10⁶ cells/1 mL of cell IMDM complete media by placing them in an incubator at 37 °C and 5% CO₂) to the viral coated wells by mixing the cell suspensions well and pipetting them into the now viral concentrated fibronectin plate. Spin at 1,200 x g at 25 °C for 90 min. Put the cells in a 37 °C, 5% CO2 incubator.
   5. 48 h post transduction, pellet the cells by centrifugation at 1,200 x g at 4 °C for 5 min and resuspend them in FACS buffer #1 (1x PBS + 0.5% BSA) to 6 x 10⁶/mL. Determine the percentage of BCR-ABL1 positive cells by measuring the percentage of YFP positive using flow cytometry.
   6. Acquire events with the standard procedure. First, gate the live cells based on forward and side scatter. Then, gate the YFP positive live cells excluding the YFP-negative cells determined by negative control (Figure 1).

Figure 1: FACS showing the frequency of YFP+ cells from the blood of a transplanted and a WT mouse (negative control). The top panels show a scatter plot of the cells and the gating. The lower panels show histograms of YFP vs. side scatter. Cells with an MFI of >10³ were considered YFP+. Similar gating was used for bone marrow cells after transduction for calculating the percentage transduction. Please click here to view a larger version of this figure.