Transient transfection protocol for the ecotropic/amphotropic producer line from Gary Nolan including titering and concentration of viral stocks.

8.0 g NaCl

6.5 g HEPES (Sigma H-7006)

up to 500 ml Q, pH to 7.0 with HCl

(initially, make pH 6.95, 7.0, 7.05 and compare side by side in transfection)

filter sterilize and store in aliquotes at -20°

up to 100 ml with Q

2.57 g chloroquine (Sigma #C6628)

up to 100 ml with Q

up to 10 ml with Q

5 mg diptheria toxin (Sigma)

up to 5 ml with 1X PBS

up to 3.3 ml with 1X PBS

filter sterilize and store in aliquotes at -20°

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

use immediately

(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

0.75 g NBT (Sigma #N-6639)

up to 10 ml with 70% DMF

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

transfection

• Plate rapidly growing freshly thawed Pheonix cells at 5-6 million per 10 cm dish (4 dishes per viral construct) at approximately noon.

• On the following morning remove the medium and add 6 ml medium containing 4 microliters chloroquine (2000X stock of 50 mM). Hold at room temperature while the DNA is prepared. For each construct approximately 24 ml medium and 16 microliters chloroquine.

• In the hood, add 4 ml 2X HBS and bubble for 15 seconds. Then drop 2 ml onto each plate and swirl several times. Repeat for each construct.

• 24 hours later remove the media and add 5 ml fresh medium.

• 24 hours later harvest the first viral supernate and store at -80°. Repeat this the following day for a total of 40 ml viral supernate.

• 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.

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) 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°.

titering