Generating Viral Stocks

In order to generate viral stocks, the viral recombinant genomes will have to be transfected either in CEFs (46) or in T2ECs. Only this second technique will be described here.

1. Prepare the following solutions in 2 Eppendorf tubes: Solution A. 25 pL of LM23 (serum-free antibiotics-free medium, see Subheading 2.) plus 4 pL of Lipofectamine™; and Solution B. 25 pL of LM23 plus 0.7 pg of endotoxin-free plasmid DNA (we routinely use the EndoFree™ Plasmid Kit from Qiagen to prepare our plasmid solutions). In the case where a defective genome has to be cotransfected with a helper genome, use a defective/helper ratio of 5:10, for a total amount of DNA of no more than 1 pg.

2. Combine solutions A and B together, mix gently, and incubate at room temperature for 25 min.

3. Centrifuge exponentially growing T2ECs (use a more than a 1-wk-old culture), resuspend them under LM23, number the cells, adjust the concentration to 10 x 106 cells/mL. Seed 200 pL of cells/well of a 24-well dish.

4. Add gently the DNA/Lipofectamine™ mixture to the cells. Incubate at 37°C for 1 h.

5. Add 250 pL of LM23 plus 20% FBS. Incubate overnight.

6. Start the applicable selection (see Note 2). If no selection is applicable, grow the cells for about 10 d, at which time the infection should be maximal. During that time period, cells should grow normally and, therefore, should be diluted every other day or so. Verify the expression of your protein under scrutiny by conventional methods.

7. Rescue supernatant by centrifugation at 1500g for 15 min, sterilize using a 0.22-pm filter, store aliquoted at -80°C. Do not reuse a thawed vial (see Note 3).

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