Viral Packaging Protocol

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Day 0: Coat 15-cm dishes with 50 µg/ml poly-d-lysine dissolved in PBS (12 ml/dish) for a period of 1 hour. Wash twice with PBS and then dispense 8–10 million 293 T cells in standard DMEM (high glucose) + 10% FBS to a total volume of 20 ml.

Day 1: After 24 h the plates should be about 70–80% confluent. Proceed to make the transfection cocktail: this modified formula (to reduce Lipofectamine and DNA amounts) for cells seeded in a 15-cm plate is as follows -

  1. Add 36 μl of Lipofectamine to 1.2 ml of OPTI-MEM (per 15 cm dish) in a 15-ml polystyrene tube, and incubate for 5 min at room temperature.
  2. In parallel, mix 24 μg of total DNA (per 15 cm dish) i.e., 3 μg VSV.G/PMD2.G, 12 μg of lenti-gag/pol/PCMVR8.2, and 9 μg of lentiviral vector (Note: all in Dongxin's maxi-prep box), into 1.2 ml of OPTI-MEM in another 15 ml polystyrene tube. After 5 min, mix the diluted DNA with diluted Lipofectamine and incubate for 30 min at room temperature.
  3. In the mean time, change to fresh media in the 293 T cultures now using DMEM (with 1%FBS) to a total volume of 20 ml.
  4. Finally, add the DNA–lipid complexes drop wise onto the 293T cultures. Gently swirl the plates and shake back and forth and sideways to mix uniformly and place dishes in the incubator. Culture overnight.

Day 3: After 48 hours, Harvest the supernatant from the plates in 50-ml tubes and store at 4°C. Add fresh DMEM with 1% FBS to a total volume of 20-ml to the plates for another harvest later.

Day 4: Harvest the second round of supernatant and collect into the original 50-ml tubes. There should now be a total of about 40 ml supernatant per each vector. Typical titers of unconcentrated viruses obtained using this procedure are in the range of ∼10^ to 5 × 10^5 transducing units/ml. To eliminate cell contamination, filter the supernatant using a 0.45-μm filter unit (low protein binding). Filters can be found on the shelf to the left of the geldoc. You will typically need about 3 filters per library.

Day 4 (part 2): To concentrate viruses by 50–100-folds the Centricon (Plus-20, 20 ml from Millipore) with a cutoff 100,000 NMWL is my method of choice. The filter filtration method also helps to reduce the free form of VSV.G proteins that are toxic to target cells. Each device concentrates ∼18 ml to ∼200 μl each time with a spin for 20–30 min at 3,000 rpm, and repeated loading is fine. Use two concentrators per library. Using this procedure starting from a volume of ∼40 ml of supernatant I typically concentrate down to about 300–500 μl. I recommend the use of freshly made viruses for transduction, but if it is not possible, make small aliquots of 150 uL and store them at −80°C where it is stable for many years.