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AAV Production and Transfection

Step1: Transfecting the HEK293T Cells

Note: Although a variety of transfection protocols may be successfully used with these vectors, the following calcium phosphate-based protocol is recommended. This protocol consistently results in the production of titers >107 viral particles/ml when transfecting the HEK293T cells.
Notes: Do not allow the transfection mixture prepared in this section to sit before it is added to the cells. The large aggregates that form after prolonged incubation are inhibitory to uptake.
The use of lipid-based transfection reagents is not recommended.
1. Inspect the host cells that were split two days before; they should be approximately 70–80% confluent.
2. Remove the three plasmids to be co-transfected (the recombinant pAAV expression plasmid or control plasmid, pAAV-RC, and pHelper) from storage at –20°C. Adjust the concentration of each plasmid to 1 mg/ml in TE buffer, pH 7.5.
3. Pipet 10 μl of each of the three plasmid DNA solutions (10 μg of each plasmid) into a 15-ml conical tube. Add 1 ml of 0.3 M CaCl2 and mix gently.
4. Pipet 1 ml of 2 × HBS (see Preparation of Media and Reagents) into a second 15-ml conical tube. Add the 1.03-ml DNA/CaCl2 mixture (see above) dropwise. Mix gently by inversion or by repeated pipetting.
5. Immediately apply the DNA/CaCl2/HBS suspension to the plate of cells in a dropwise fashion, swirling gently to distribute the DNA suspension evenly in the medium.
6. Return the tissue culture plate to the 37°C incubator for 6 hours.
7. At the end of the incubation period, remove the medium from the plate and replace it with 10 ml of fresh DMEM growth medium.
8. Return the plate to the 37°C incubator for an additional 66 hours.

Step2: Preparing Viral Stocks

Note: Viral particles are present in both intact cells and the growth medium. Preparation of the viral stock from the combined suspension of cells plus growth medium results in the greatest yield of virus. If a more concentrated virus stock is required, see references 1, 4, 5 and 61, 4, 5, 6for virus stock concentration and purification protocols.
1. Prepare a dry ice-ethanol bath and a 37°C water bath.
2. Transfer the transfected cells plus DMEM growth medium to a 15-ml conical tube. To collect the cells from the plate, scrape the cells into the pool of growth medium with a cell lifter, while holding the plate at an angle.
3. Subject the cell suspension to four rounds of freeze/thaw by alternating the tubes between the dry ice-ethanol bath and the 37°C water bath, vortexing briefly after each thaw. Note Each freeze and each thaw will require approximately 10 minutes’ incubation time.
4. Collect cellular debris by centrifugation at 10,000 × g for 10 minutes at room temperature.
5. Transfer the supernatant (primary virus stock) to a fresh tube. Viral stocks can be stored for more than one year at –80°C.

Step3: Viral Titer Measurement

Culture the cells with 5 ml viral stock and 1 ml DMEM growth medium for 1day and replace it with 5 ml of fresh DMEM growth medium. Measure the positive rate with flow cytometry.

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