Figure 4.1-B. Evaluation of the replication potential of AvlCFl DNA in HeLa 
cells by 32 P- labelling of DNA after infection of HeLa cells by AvlCFl. HeLa 
cells were grown to 80% confluency in Dulbecco's modified Eagle medium 
(DMEM) , 10% fetal calf serum, 1% glutamine, 100 U/ml penicillin, 100 /xg/ml 
streptomycin and infected with AvlCFl at a multiplicity of infection (MOI) 
of 10 to 1000 plaque forming units (pfu) per cell (Appendix 1) . After 20 
hr, the media was replaced with phosphate -free DMEM containing 32 P0*“ 
(Berkner and Sharp, 1983) as well as supplements described above. Following 
a 20 hr labeling period, cells were lysed and viral DNA was prepared by the 
Hirt extraction method (Hirt, 1967). The DNA was purified by proteinase K 
treatment and phenol extraction, cleaved with EcoRI and evaluated by aga- 
rose gel electrophoresis. Lane 1- uninfected control (note the doublet of 
mitochondrial DNA). Lane 2- positive control infection with Ad5. Lane 3- 
AvlCFl lot A7-3, MOI 10; Lane 4-6, same as lane 1 but at MOI of 30, 100, 
1000 respectively. Lanes 7-10, same as lanes 3-6, but with AvlCFl lot A7-5. 
No AvlCFl DNA replication was observed at MOI of 10 or 30 , but at higher 
MOI (100, 1000), HeLa will support AvlCFl replication. 
Unin- 
fected Ad5 
Multiplicity f ~ ~ 
of infection \ 
Ad5 specific -► m 
AvlCFl 
Lot A7-3 Lot A7-5 
AvlCFl - 
specific 
12 3456789 10 
Recombinant DNA Research, Volume 16 
[691] 
