Figure 4.1-C. Evaluation of the replication potential of AvlCFl DNA in 
freshly isolated normal human bronchial epithelial (HBE) cells. The repli- 
cation potential of AvlCFl in HBE cells was evaluated by 32 PC> 4 ,“ labeling of 
nascent DNA after infection of HBE cells by the recombinant adenovirus 
AvlCFl. Human bronchial epithelial cells were obtained at bronchoscopy 
using a standard cytology brush (Trapnell et al. , 1991a) and were cultured 
in suspension in DMEM, 10% fetal bovine serum, 1% glutamine, 100 U/ml 
penicillin, 100 /xg/ml streptomycin and infected with AvlCFl at a MOI of 100 
pfu/cell for 48 or 72 hr, or at various MOIs (10, 100, 500, 1000 pfu/cell) 
for 48 hrs . The media was replaced with phosphate -free DMEM containing 
inorganic 32 P0 A = and the supplements described above. Following a 20 hr 
labeling period, cells were lysed and viral DNA was prepared by the Hirt 
extraction method (Hirt, 1967). DNA was purified by proteinase K treatment 
and phenol extraction, cleaved with EcoRI and evaluated by agarose gel 
electrophoresis. No labeled viral DNA is seen in control, uninfected cells 
(lane 1). As expected, infection by Ad5 (MOI = 100) resulted in 32 P-labeled 
viral DNA restriction fragments of the sizes expected for Ad5 after 48 
(lane 2) and 72 (lane 3) hr. In contrast, infection by AvlCFl (MOI = 100) 
did not generate labeled DNA fragments at 48 hr (lane 4) or 72 hr (lane 5) 
indicating a lack of replication of AvlCFl DNA in HBE cells. As a positive 
control for the function of AvlCFl, AvlCFl infected cells showed CFTR 
protein detected by immunohistochemistry (not shown) . As a positive control 
for the assay, infection of HBE cells with Ad5 MOIs ranging from 10 to 1000 
showed 32 P-labeled DNA restriction fragments of Ad5 (lanes 7-10) indicating 
replication of Ad5 in HBE cells. In contrast, infection of HBE cells with 
AvlCFl at MOIs ranging from 10 to 1000 showed no labeled DNA fragments 
(lanes 11-14) indicating no replication of AvlCFl DNA in HBE cells. 
Recombinant DNA Research, Volume 16 
[693] 
