Purification of Deletion Mutants From Mixed Stocks

Because adenovirus particles with differing DNA contents have differing buoyant densities in CsCl, adenovirus deletion mutants grown in the presence of helper virus can be separated from the helper by equilibrium sedimentation in CsCl density gradients. Only mutants with fairly large deletion mutations (>10%) can be efficiently purified from helpers with a wild-type genome size by this method, although helper virus with longer than wild-type genomes have been used to make the purification of mutants with smaller deletions possible (19).

1. Prepare a mixed lysate. One dish should be labeled with 32P, as described below.

2. To the mixed stock, add IGEPAL to a final concentration of 0.05%.

3. Extract the stock vigorously with 1/5 vol of 1,1,2-trichlorotrifluoroethane. Recover the aqueous phase after centrifugation at 4000g for 5 min in a Sorvall GSA rotor. Re-extract the cell debris and organic phase with a small volume of PBS; recover the aqueous phase and pool with the supernatant from the previous centrifugation.

4. Centrifuge the pooled supernatants at 10,000 rpm (g) for 5 min (Sorvall SS34 rotor) to remove small particulate cell debris.

5. Prepare a discontinuous CsCl gradient in a 35-mL polypropylene centrifuge tube by adding (in order) approx 20 mL extracted virus suspension, 4 mL of CsCl density 1.25, and 5 mL of CsCl density 1.7. Add each solution slowly through a pipet placed all of the way to the bottom of the tube. After the CsCl solutions have been added, fill the tube to the desired level with extracted virus suspension.

6. Centrifuge for 90 min at 29,000g (Sorvall SV288 rotor or equivalent) or 3 h at 82,000g (Beckman SW27 rotor or equivalent).

7. In a darkened tissue culture hood, illuminate the gradient with narrow beam of light from one side. A microscope lamp is a suitable light source. The virus will form a sharp, blue-white, translucent band at the interface of the two CsCl solutions. A broader, yellowish or tan, frequently granular layer of cell debris will appear at the top of the lighter CsCl cushion.

8. Collect the virus, avoiding the cell debris (see Note 3).

9. Adjust the concentrated virus suspension to a density of 1.34 (refractive index 1.3663) with 20 mM Tris-HCl, pH 7.5, or with the density 1.7 CsCl solution. Place in a centrifuge tube and fill to the required volume with CsCl density 1.34.

10. Centrifuge the suspension at 35,000 rpm (g) for 16 h in a Sorvall TV865 rotor (or equivalent). Two closely spaced virus bands should be visible in the center of the tube.

11. Fractionate the gradient into single-drop fractions through a hole made in the bottom of the tube.

12. Measure the radioactivity in each fraction by Cherenkov counting. Two more or less well-separated peaks should appear (Fig. 1).

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