Viral-Mediated Gene Transfer to Study the Behavioral Correlates of CREB Function in the Nucleus Accumbens of Rats - Drugs of Abuse: Neurological Reviews and Protocols - page 329

of OptiMEM in a second tube, and then add it to the 250- L DNA mixture in

the fi rst tube. Leave the DNA-LipofectAMINE mixture at RT for 20-45 min,
for liposomes to form.

µ

4. Remove the medium from the plates and wash them once with 2 mL of OptiMEM,
remove, and replace with 2 mL of OptiMEM.

5. Add the DNA-LipofectAMINE mix to the cells dropwise evenly over the whole
plate. Incubate at 37°C for 5-7 h.

6. Prewarm D-PBS + Ca 2+ , Mg 2+ and DMEM + 10% FBS to 37°C (about 20 min).

7. Wash the cells three times with 2 mL of D-PBS + Ca 2+ , Mg 2+ . Replace medium
with 3 mL DMEM + 10% FBS. Incubate cells overnight at 37°C.

3.1.2. Superinfect Transfected Cells—P0

1. Allow the cells to recover at least 20 h from the time of the last wash after the
transfection. Prewarm DMEM + 5% FBS to 37°C (about 20 min).

2. Remove the medium from the plates and add 5 mL of DMEM + 5% FBS. Add
approx 6

10 5 pfu of helper virus and incubate at 37°C until 95% of the cells
show cytopathic effects (CPE) (have rounded up). This will take 30-40 h.

×

3.1.3. Harvest Cells—P0

1. Harvest the cells by pipetting the medium onto them until they have all detached
from the plate or scraping them up with a cell lifter, and transfer cells and
medium to 15-mL polypropylene conical tubes with plug seals.

2. Freeze-thaw the cells three times using a dry ice-ethanol bath and a 37°C water
bath. Minimize the amount of time that they are thawed in the 37°C bath.

3. Transfer the cells to 15-mL polystyrene tubes and sonicate them for 2 min in
a cup sonicator (power setting 6, 50% duty cycle, 1-s cycles). Centrifuge the
cells for 5 min at low speed (1350 g for 5 min) to pellet cell debris but not virus
particles. Plate the supernatant on cells for the P1 passage or store it at -70°C.

3.1.4. Amplify Virus Stock—P1

10 5 cells per 60-mm dish in 5 mL of DMEM + 10% FBS.
Incubate at 37°C for 2 d.

1. Plate fresh 2-2 cells: 4 ×

2. Replace the medium with 4 mL of DMEM + 2% FBS and add 4 mL of the P0
supernatant.

3. Harvest and process the cells as in Subheading 3.1.3. when they show 95%
CPE (about 24 h).

3.1.5. Amplify Virus Stock—P2

1. Plate fresh 2-2 cells: 1 × 10 6 per 100-mm dish in 10 mL of DMEM + 10% FBS

(two dishes per sample).

2. Two days later, replace the medium in each dish with 6.0 mL of DMEM + 5% FBS
and add 4.0 mL of P1 supernatant per dish.

3. Incubate the cells at 37°C overnight, and process them as described in Subhead-
ing 3.1.3. (but using 50-mL instead of 15-mL conical tubes) when they show
95% CPE (about 24 h).