Homologous Recombination

1. Day 1: Initiate culture of CV-1 cells. Seed a T-25 flask (one flask per construct) with CV-1 cells at 1 x 106 cells/flask, in a total volume of 10 mL of DMEM + 10% FCS. Use the next day when ~80% confluent (should be ~3 x 106 cells/flask).

2. Day 2: Infection and transfection. Thaw WR virus stock (titer of stock should be ~5 x 109 pfu/mL). Sonicate for 30 s, chill on ice for 2 min, and vortex vigorously for 30 s. Repeat once.

3. Trypsinize virus by mixing 20 ^L of virus with an equal volume of trypsin (cell culture grade). Incubate at 37°C for 30 min, vortexing briefly every 5 min. This is tube A, a 1/1 dilution (~2.5 x 109 pfu/mL).

4. Dilute A 1/100 in 2.5% DMEM (10 ^L A + 990 ^L 2.5% DMEM). This is tube B, a 1/200 dilution (~2.5 x 107 pfu/mL).

5. Aliquot a volume of B containing 1.5 x 105 pfu (~6 ^L) and add to 1 mL of 2.5% DMEM. This is tube C, final concentration of 1.5 x 105 pfu/mL.

6. Aspirate medium from the T-25 flask of CV-1 cells, and replace with 1 mL of tube C (represents ~0.05 pfu per cell).

7. Incubate for 2 h at 37°C in 5% CO2, with gentle rocking every 15 min.

8. Set up transfection reagents 30 min before end of step 7. Note that all plasmids should be at 1 ^g/^L, and lipofectin is inhibited by serum proteins and penicillin.

9. Set up two sterile polystyrene tubes:

b. Tube B: 30 ^Llipofectin (or lipofectamine) + 200 ^L DMEM SF

10. Gently mix A and B. Incubate for 30 min at room temperature. Add 1.6 mL of DMEM SF to the AB mixture.

11. Aspirate the virus inoculum from the T-25 flask. Add the DNA/lipofectin transfection mixture (volume 2 mL) to the cells, ensuring good coverage of the cell layer.

12. Incubate for 5-6 h in a cell culture incubator.

13. Overlay transfection with 3 mL of 10% DMEM (do not aspirate transfection cocktail). Continue culture for 2 d.

14. Day 4: Harvest homologous recombination. Cytopathic effect (CPE) should be clearly visible by light microscopy. Scrape cell layer with a rubber policeman into a 15 mL conical tube. Pellet cells by centrifugation at 300g (3000 rpm), 10 min in a bench-top centrifuge. Aspirate and discard supernatant. Resuspend pellet in 0.5 mL of 2.5% DMEM.

15. Freeze and thaw lysate three times in a Dry Ice/ethanol bath.

16. Store at -80°C, or proceed with plaque assay (see Subheading 3.2.2.).

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