Figure 4.1-D. Evaluation of the replication potential of AdCFTR DNA in 
freshly isolated normal human bronchial epithelial (HBE) cells. The repli- 
cation potential of AdCFTR in HBE cells was evaluated by 32 P0 4 “ labeling of 
nascent DNA after infection of HBE cells by the recombinant adenovirus 
AdCFTR. Human bronchial epithelial cells were obtained at bronchoscopy 
using a standard cytology brush (Trapnell et al., 1991a) and were cultured 
in suspension in DMEM, 10X fetal bovine serum, IX glutamine, 100 u/ml 
penicillin, 100 pg/ml streptomycin, 2.5 pg/ml Fungizone and infected with 
AdCFTR 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 PO 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 evalu- 
ated by agarose gel electrophoresis. As a positive control for viral DNA 
replication, 293 cells were infected with Ad5 or AdCFTR (10 pfu/cell), 
labeled simultaneously with 32 P0<, = for 16 hr, and then processed as above. 
No viral DNA is seen in control uninfected 293 cells (lane 1) . As expected, 
infection with Ad5 resulted in three 32 P- labeled viral DNA restriction 
fragments of the predicted sizes for Ad5 (lane 2 ; 27 kb, 5 . 6 kb , and 2.7 
kb). Infection by AdCFTR resulted in four 32 P-labeled viral DNA restriction 
fragments of the predicted sizes for AdCFTR (lane 3; 25 kb, 6.4 kb, 3.3 kb 
and 1.5 kb). The doublet of mitochondrial DNA is indicated by "M" . No 
labeled viral DNA is seen in control, uninfected HBE cells (lane 4). 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 5) and 
72 (lane 6) hr. In contrast, infection by AdCFTR (MOI = 100) did not gener- 
ate labeled DNA fragments at 48 hr (lane 7) or 72 hr (lane 8) indicating a 
lack of replication of AdCFTR DNA in HBE cells. As a positive control for 
the assay in HBE cells, infection of HBE cells with Ad5 at MOIs ranging 
from 10 to 1000 showed 32 P-labeled DNA restriction fragments of Ad5 (lanes 
9-13) indicating replication of Ad5 in HBE cells. In contrast, Infection of 
HBE cells with AdCFTR at MOIs ranging from 10 to 1000 showed no labeled DNA 
fragments (lanes 14-17) indicating no replication of AdCFTR DNA in HBE 
cells . 
Recombinant DNA Research, Volume 16 
[695] 
