M.J. Welsh and A.E. Smith, RAC Application 
Mixing experiments adding wild-type Ad2 sequences to preparations of El-deleted virus 
with primer set 2 indicate that we can detect 10 molecules of wild-type DNA in the 
presence of 10 9 particles of El -deficient virus. 
Concentration - The total particle concentration of the viral seed stock will be measured by 
absorbance at 260 nm (A 260 X where 1.0 OD (A 26 O) = 1.1 x 10 12 particles. 
Activity - The infectious unit concentration of the viral seed stock will be measured by end- 
point CPE titration assays and verified using immunofluorescence using FITC-conjugated 
Ad antibody (Chemicon). 
9. Growth of Production Lots of Virus 
Production lots of Ad2/CFTR- 1 will be produced by inoculation of approximately 5 to 
10 x 10 7 PFU of MVSS onto approximately 1-2 x 10 7 WCB 293 cells grown in a T175 
flask containing 25 mis of 925 medium. Inoculation is achieved by direct addition of the 
virus (approximately 2-5 mis) to each flask. Batches of 50-60 flasks constitute a lot. 
Following 40-48 hours incubation at 37°C, the cells are shaken loose from the flask and 
transferred with medium to a 250 ml centrifuge bottle and spun at 1000 xg. The cell pellet 
is resuspended in 4 ml phosphate buffered saline containing 0.1 g/1 CaCl 2 and 0.1 g/1 
MgCl2- The protease inhibitors aprotinin (0.1 |ig/ml) and leupeptin (0.5 fig/ml) are added 
and the cells subjected to two cycles of freeze-thaw to release virus. Cellular debris is 
removed by centrifugation at 1000 xg for 15 min. The supernatant from this centrifugation 
is layered on top of the CsCl step gradient: 2 ml 1.4 g/ml CsCl and 3 ml 1.25 g/ml CsCl in 
10 mM Tris, 1 mM EDTA (TE) and spun for 1 hour at 35000 rpm in a Beckman SW41 
rotor. Virus is then removed from the interface between the two CsCl layers, mixed with 
1.35 g/ml CsCl in TE and then subjected to a 2.5 hour equilibrium centrifugation at 75,000 
rpm in a TLN-100 rotor. Virus is removed by puncturing the side of the tube with a 
hypodermic needle and gently removing the banded virus. To reduce the CsCl 
concentration, the sample is dialyzed against 2 changes of 2 liters of Tris buffered saline. 
Following this procedure, dialyzed virus is stable at 4°C for a few days or can be stored for 
longer periods at -80°C. Aliquots of material for human use will be tested and while 
awaiting the results of these tests, the remainder will be stored frozen. The tests to be 
performed are described below: 
10. Tests for Virus Productions Lots (to be performed by Genzyme) 
Identity - Restriction map and SDS PAGE, as described for MVSS. 
Purity - Absence of wild-type virus by El PCR, as described for MVSS. The virus 
production lot will be evaluated for the presence of any inapparent transforming factors or 
agents by cultivation on Rat 1 indicator cells (details below). The virus lot will be 
evaluated for the presence of residual bovine serum proteins with either a Western blot or 
ELISA. 
Concentration - A 26 O 1 as described above. 
Activity - End-point CPE titration assays. From the activity and concentration 
measurements, we can calculate a particles per infectious unit (I.U.) ratio (I.U. as 
determined by end point CPE and PFU are related by a factor of 0.7). Typically the 
particle/PFU ratio is 350-750, only preparations with a ratio less than 500 will be used. 
[880] 
Recombinant DNA Research, Volume 16 
