are human embryonic kidney cells that have been transformed into a continu- 
ous line by Ad5 ; the genome of 293 cells include 11% of the left end of the 
Ad5 genome (0 to 11 m.u.) (Graham et al., 1977; Precious and Russell, 
1985) . This region provides El functions in trans that have been deleted 
from the genome of AdCFTR and AvlCFl , and permits production of replication 
deficient, recombinant infectious virions of AdCFTR and AvlCFl. The 293 
cells were transfected with the purified Clal cut genome from Addl 327 
together with the plasmid DNA for AdCFTR or AvlCFl and then cultured in a 
humidified atmosphere at 37* with 5% C0 2 . After 18 hours, the cells were 
washed and 3 ml of soft agarose media (minimal essential media containing 
1% seaplaque agarose and 7.5% fetal calf serum) was added. Plaques were 
picked after 15 days and amplified on 293 cells in successively larger 
plates usirg standard methods (Green et al . , 1967; see Appendix 1 for 
further details) . 
High titers of the AdCFTR and AvlCFl recombinant adenovirus vector were 
produced in 293 cells and purified on cesium chloride gradients. To accom- 
plish this, cells were grown to 70-80% confluency and infected with puri- 
fied AdCFTR or AvlCFl virus at a multiplicity of infection of 10 pfu/cell 
and cultured until the cells showed typical cytopathic effect. The cells 
were then harvested and lysed by freezing and thawing to release intracel- 
lular virus . The crude viral lysate was cleared of cellular debris by low 
speed centrifugation. Infectious virus was then recovered by centrifugation 
on a two step cesium chloride gradient to remove low molecular weight 
cytoplasmic contents and empty viral capsids (Precious and Russell, 1985). 
The virus was further purified by isopycnic density centrifugation on 
cesium chloride. Cesium chloride was removed by dialysis and the resultant 
virus was aliquoted and stored at -70* until use. The final viral prepara- 
tion included 10% glycerol to stabilize the virus during storage. 
The amount of infectious viral particles in all preparations was determined 
by plaque assay to titer adenovirus (Green et al . , 1967). The plaque assay 
was carried out with serial dilutions of the virus used to infect 75% 
confluent 293 cells cultured in 6 well plates. After 90 minutes, the media 
was changed to soft agarose media and the cultures were continued in a 
humidified environment at 37* in 5% C0 2 and observed for the appearance of 
plaques. Titers were determined by counting viral plaques at 2 weeks. 
2.5 Formulation and Quality Control Parameters 
The final production of the AdCFTR and AvlCFl vectors involves: (1) purifi- 
cation of virions away from the intracellular debris of the transfected 293 
cells by low speed centrifugation; (2) CsCl step gradients to remove low 
molecular weight cellular and unincorporated viral protein components; (3) 
CsCl isopycnic density gradient centrifugation to remove incomplete virions 
and empty viral capsids (all have lower density than infectious adenovi- 
rus); and (4) dialysis to remove low molecular weight components, including 
the CsCl used for the gradient centrifugation. To maintain stable, intact, 
infectious recombinant adenovirus during and subsequent to dialysis, the 
final dialysate includes 10 mM Tris-Cl, pH 7.4, 1 mM Mg Cl 2 , and 10% glyc- 
erol (to prevent aggregation of virions). 
Recombinant DNA Research, Volume 16 
[667] 
