Editing Adenovirus pAd52 (section)

== Production and Purification of High Titer Adenovirus Stock ==

General comments. Many experimental studies can be carried out using virus in the form of crude infected cell lysates prepared as described above. However, for some experiments, particularly for animal work it is usually desirable to use purified virus. The following protocol is adapted from Hitt et al. (1998).

Recombinant Ads can be purified from crude lysates of either monolayer or suspension cultures. Suspension cultures are easier to harvest, but it is easier to determine the correct time to harvest (i.e., complete or near complete cpe) when using monolayer cultures. A subline of 293 cells, 293N3S cells, adapted to growth in suspension is available from Microbix. The following protocol describes a method for banding virus obtained from 3-liters of infected suspension culture, or about 30 x 150-mm dishes.

Protocol<br>1. Prepare crude cell lysate from infected cells:

a) For 3-liter spinner cultures: when inclusion bodies are visible in 80-90% of the cells, harvest by centrifugation at 750 x g for 20 min in sterile 1-liter bottles. Combine pellets in a small volume of medium, and spin again.

Resuspend pellet in 15-30 ml 0.1M Tris-HCl, pH 8.0: if wild-type levels of virus are expected, resuspend in 30 ml, if lower yields are expected (some vectors may replicate to lower yields than others), use 15 ml. Store sample at -70oC.

b) For 30 x 150-mm dishes: when all infected cells are rounded and most are floating, scrape dishes, then transfer cells and supernatant to 50-ml centrifuge tubes and spin 10 min at 750 x g. Resuspend cell pellet in 15 ml 0.1M Tris-Cl, pH 8.0. Store sample at -70oC.

2. Thaw sample and add 1.5 ml 5% Na deoxycholate for each 15 ml of cell lysate. Mix well and incubate at room temperature for 30 min. This results in a relatively clear, highly viscous suspension.

3. Add 150 μl 2 M MgCl2 and 75 μl DNase I solution for each 15 ml of cell lysate, then mix well. Incubate at 37oC for 30-60 min, mixing every 10 min. The viscosity should be reduced to only slightly more than that of water.

4. Spin 15 min, top speed, at 5oC in the Beckman table top centrifuge.

5. Meanwhile, prepare CsCl step gradients (one SW41 ultraclear tube for each 5 ml of sample) in the virus hood: Add 0.5 ml of 1.5g/cc solution to each tube. Gently overlay with 3.0 ml of 1.35g/cc solution. Gently overlay this with 3.0 ml of 1.25g/cc solution. Do not disturb gradients once they are formed.

6. Apply 5 ml of supernatant from step 4 to the top of each gradient. If necessary, top up tubes with 0.1M Tris-Cl, pH 8.

7. Spin at 35K rpm in SW41 rotor, 10 oC, for 1 hour (accelerate/decelerate set to 1).

8. Collect the viral bands (should be at the 1.25g/cc-1.35g/cc interface) and pool. (One can collect by sealing the top of the tube with a piece of parafilm, puncturing the parafilm with a needle, then puncturing the bottom of the tube with a red hot 16 gauge needle, controlling the flow of solution out the bottom with a gloved finger over the top hole.)

9. Transfer the pooled virus to a SW50.1 ultraclear tube (using the 1.35g/cc CsCl solution to top up the tube), mix well, and centrifuge in the SW50.1 rotor at 35K rpm, 4oC, for 16-20 hr. (Alternatively, the pooled virus can be centrifuged in the SW41 rotor at 35K rpm, 10oC, 16-24 hr.)

10. Collect the virus band in the smallest volume possible (usually 0.5 to 1 ml), transfer to a Slide-A-Lyzer dialysis cassette, and dialyze at 4oC against 3 changes of 500 vol (or more) 10 mM Tris-HCl, pH 8.0, for at least 24 hr total.

11. After dialysis, add sterile glycerol to a final concentration of 10%. Store the purified virus at -70oC in small aliquots.

Materials

The following special equipment is required:<br>Beckman SW 41 rotor,<br>SW50.1 rotor, ultraclear tubes for each rotor,<br>Pierce Slide-A-Lyzer dialysis cassettes, sterile 1-liter beaker with stir bar, syringes and needles.

Solutions<br>1. 0.1 M Tris-HCl, pH 8.0: Add 1.2 g Tris base to 80 ml H2O. Adjust pH to 8.0 with HCl. Adjust volume to 100 ml and autoclave.

2. 5% Na deoxycholate: Add 5 g Na deoxycholate to 100 ml H2O.

3. 2M MgCl2: Add 40.6 g MgCl2.6H2O to 100 ml H2O and sterile filter.

4. DNase I solution: Dissolve 100 mg bovine pancreatic deoxyribonuclease I (DNase I) in 10 ml of 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 50% glycerol. Store in small aliquots at -20oC.

5. CsCl solutions for banding:<br>density CsCl 10 mM Tris, pH8<br>1.50 g/cc 90.8 g 109.2 g<br>1.35 g/cc 70.4 g 129.6 g<br>1.25 g/cc 54.0 g 146.0 g

Add 10 mM Tris to indicated amount of CsCl and stir to dissolve. Verify density by weighing 1 ml of each solution. Sterile filter and store at room temperature.

6. Dialysis buffer: 10 mM Tris, pH 8.0. Use at 4oC.

7. Sterile glycerol: Prepare by autoclaving.

<br><br>

[[Category:Protocols]]