This transient transfection protocol for the ecotropic/amphotropic producer line from Gary Nolan includes instructions for titering and concentration of viral stocks. I have found that this procedure gives consistently higher transfection efficiency than the protocol from the Nolan Lab.

50 mM HEPES 47.64 g HEPES (Sigma #H9136)

280 mM NaCl 65.45 g NaCl

pH to 7.1-7.3 with approximately 8 ml

10N NaOH, bring up to 4 liters with Q

and autoclave

up to 400 ml with Q

70 mM sodium phosphate 0.966 g NaH₂PO₄-H₂O

up to 100 ml with Q, autoclave

high molecular weight calf thymus DNA

Boehringer Mannheim #104-167

bring up to 1 mg/ml with sterile Q

store at 4°, do not freeze

2.57 g chloroquine (Sigma #C6628)

up to 100 ml with Q

up to 10 ml with Q

up to 5 ml with 1X PBS

1 g hygromycin (Calibochem)

up to 3.3 ml with 1X PBS

2 g KCl

up to 1 liter with Q, pH to 7.4 and autoclave

200 g paraformaldehyde

1 ml 10N NaOH

up to 1 liter with Q, heat to 65° to dissolve, aliquote and store at -20°

mix 100 ml 20% paraformaldehyde with 50 ml 10X PBS and bring up to 500 ml with Q

filter, and store at 4° for up to 2 weeks

(Nolan says to use 0.05% gluteraldehyde to fix 3T3 for titering)

25 ml 1X PBS

(for every 7.5 ml add 25 microliters NBT and 19 microliters BCIP)

50 ml 2M Tris 9.5

up to 1 liter with Q

0.5 g BCIP (p-toluine salt Sigma #B-8503)

up to 10 ml with DMF

store at -20° in light proof glass container

0.75 g NBT (Sigma #N-6639)

up to 10 ml with 70% DMF

store at -20° in light proof glass container

14.7 g Potassium Ferrocyanide 35 mM (Sigma #P-9387)

11.5 g Potassium Ferricyanide 35 mM (Sigma #P-8131)

0.02% NP40 (diluted from a 10% stock)

0.01% Deoxycholic Acid Sodium Salt (Sigma #D-6750)

50 ml 10X PBS

up to 500 ml with Q

up to 10 ml in DMF

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.

concentration of virus

• 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°.

3T3 Titering

• After 48 hours stain the plates and determine the retroviral titer.