virus production
• Plate rapidly growing freshly thawed Pheonix cells at 5-6 million per 10 cm dish (4 dishes per viral construct) at approximately noon or use 1.5-2 million cells per 6 cm dish if you don’t plan to concentrate the virus.
• On the following morning remove the medium and add 3 ml medium containing 1.5 microliters chloroquine (2000X stock of 50 mM). Hold at room temperature while the DNA is prepared.
• For each construct (6 cm dish measurements) mix 7.5 micrograms of the retroviral vector, 5 micrograms of the helper construct and 20 microliters of carrier DNA. Bring up to 250 microliters with sterile Q and add 250 microliters 0.5M CaCl2. Vortex and hold at room temperature for 10 minutes.
• Mix the HNP solution by combining 1.95 ml HN buffer with 45 microliters of Phosphate Buffer.
• Put 0.5 ml HNP solution in a falcon tube and while vortexing slowly add the DNA mixture dropwise (about 1 drop per second is fine). A very fine precipitate should form. If there is large stringly precipitate then the pH is too basic and if there is no precipitate then the pH is too acidic. Hold at room temperature for 15 minutes.
• Slowly add the DNA mixture to the plate containing the Pheonix cells and swirl gently. Incubate in tissue culture incubator overnight.
• Change the medium on the next morning and transfer the plate to 32 °C for viral production. 24 and 48 hours later harvest the virus. Spin at 2,000 rpm to pellet any cells and flash freeze in liquid N2. Store at -80°C.
• Fix 1 plate from each transfection and stain to determine the transfection efficiency.
For AP vectors use 4% Paraformaldehyde for 5 minutes followed by 3 washes of 5 minutes in 1X PBS. Following the washes, heat inactivate in a 65° water bath for 45 minutes. Equilibrate in AP detection buffer and incubate in the dark at room temperature in the presence of NBT and BCIP (24 hours will give complete staining).
For X-gal staining use 0.5-0.05% Gluteraldehyde in 1X PBS for 5 minutes followed by 3 washes of 5 minutes each in 1X PBS. Following the washes add X-gal detection buffer with X-gal and incubate at 37° overnight.
• If 40-50% efficiency is achieved then proceed with the concentration and or viral overexpression analysis.
• Chill the rotor by spinning at 1000 rpm at 4° for 1-2 hours. Incubate the viral supernate at 37° until just thawed. Immediately filter through Corning 50 ml filter (0.45 micron cellulose acetate #430314) or omit this step if they have been spun down prior to freezing and put into an ultraclear tube for the SW28 rotor. Spin at 21,000 rpm for 3 hours at 4°.
• Pour off supernate and aspirate the last drop from the lip. Shake on ice for 30 minutes in the cold room. Scrape sides and pipet up and down 50-100 times to dislodge pellet of virus. Aliquote (20 microliters) and store at -80°.
