Retroviral Mediated Gene Transduction: A Gene Transfer Technique to Deliver a Transgene in Cultured Cells Using Engineered Retroviral Vectors

1. Prepare Retroviral Construct

1. Sub-clone the T-cell receptor (TCR) alpha and beta chains, separated by a 2A linker, into an MSCV-based retroviral vector (example, pMIAII or pMIGII).
   NOTE: The 2A linker allows for the stoichiometric and concordant expression of the alpha and beta TCR chains from a single open reading frame. The vector includes an IRES fluorescent protein cassette (ametrine or GFP) that is used to track transduced cells and transduction efficiency.
2. Isolate Plasmid DNA using a commercially obtained plasma prep kit or a similar product. Use a working concentration of 1-2 μg μl^-1.

2. Generation of Retroviral Producer Cell Lines

1. Utilize a two-step process to produce the retroviral producer cell lines.
   1. Generate retrovirus by transient transfection of 293T cells with three plasmids (pVSVG, pEQ-Pam3(-E) and vector of interest) and take the retroviral supernatant from the 293T cells to transduce the GP+E86 ecotropic retroviral producer cell line.
   2. Isolate the top 40% of fluorescent expressing GP+E86 producer cells by FACS and propagate the transduced-GP+E86 producer cells as a source of the virus.
      NOTE: The generation of high-titer producers is critical for the efficient transduction of bone marrow cells.

3. Retrovirus-mediated Stem Cell Gene Transfer (Day -5)

1. Thaw out and culture 0.5 -1.0 x 10⁶ of each GP+E86 producer cell line in complete DMEM 10% (vol/vol) FBS to allow sufficient growth for two confluent 150-mm plates by Day 0 of this protocol.
   NOTE: Thawing 0.5 – 1.0 x 10⁶ of each GP+E86 producer cell line in a 10 cm plate will allow for 30 – 50% confluency at Day 5. This also allows for 5 days of culture in order to expand the GP+E86 producer cell lines to two confluent 150-mm plates by Day 0.
   1. Culture retroviral producer cell line in complete tissue culture media containing 5% fetal calf serum, and ciprofloxacin (10 μg/ml) to avoid mycoplasma contamination.
      NOTE: Discontinue the use of ciprofloxacin when generating the viral supernatant for bone marrow transduction. Avoid over-growth of the producer cell line, as this will negatively impact bone marrow transduction efficiency.

4. Retroviral Producer Cell Culture (Day 0)

1. Plate 3 x 10⁶ retroviral producer cells per 150-mm plate in 18 ml complete DMEM 20% (vol/vol) FBS.
2. Incubate retroviral producer cells at 37 °C overnight. Anticipate one 150-mm plate for 15 million bone marrow cells (approximately 2-3 donor mice).

5. Retroviral Supernatant (Day 1)

1. The next day (approximately 24 hr later), harvest the retroviral supernatant from the producer plates (set up in step 4) by drawing up the retroviral supernatant directly off of the 150-mm plate with a 10-ml syringe and filter using a 0.45-μm syringe filter.
2. Carefully replace the removed retroviral supernatant with 18 ml of fresh complete DMEM 20% (vol/vol) FBS.
3. Supplement the filtered retroviral supernatant with IL-3 (20 g ml^-1), IL-6 (50 g ml^-1), MSCF (50 g ml^-1), and Hexadimethrine bromide (Polybrene) (6 μg ml^-1).
   NOTE: Hexadimethrine bromide should be made fresh every 2 months to avoid a drop in efficacy of retroviral transduction.

6. Retroviral Supernatant and Transduction (Day 2)

1. After 24 hr, collect fresh viral supernatant from the viral producer cell line. Filter the retroviral supernatant using a 10-ml syringe and a 0.45-μm syringe filter. Supplement the filtered retroviral supernatant with IL-3 (20 g ml^-1), IL-6 (50 g ml^-1) and SCF (50 g ml^-1), and Hexadimethrine bromide (6 g ml^-1).
2. Next, carefully remove with a pipette or aspirate the top 2 ml of media from each well of the six-well plate containing the bone marrow cells.
   NOTE: Work carefully not to aspirate the bone marrow, which is usually concentrated in the middle of the well. Alternatively, the removed supernatant can be spun down to recover any lost bone marrow cells.
3. To each well in the bone marrow 6-well plate add 3 ml of fresh viral supernatant containing cytokines and Hexadimethrine bromide. Wrap the plates in plastic and repeat the spin transduction at 1,000 x g for 60 min a 37 °C. Unwrap the plates and incubate cells at 37 °C and 5% CO₂ overnight (about 24 hr).

7. Addition of Fresh Supplemented DMEM (Day 3)

1. After 24 hr, remove the top 2 ml of media from each well of the six-well plate containing the bone marrow cells, as in step 6.2. Replace the removed media with fresh DMEM 20% (vol/vol) FBS supplemented with final concentrations of IL-3 (20 g ml^-1), IL-6 (50 g m^l-1), and SCF (50 g ml^-1).
2. Incubate bone marrow plates at 37 °C CO₂ incubator for another 24 hr.