Table II 
In Vitro Transfer of the E. coli Lac Z Gene by AvlLacZ4: Evaluation by X-Gal Staining 
Percent of Cells With Blue Nuclear Staining 1 
MOI = 10 pfu/cell MOI = 50 pfu/cell 
Time after infection Cell Line Cell Line 
(hr) 
H441 
WI-38 
A549 
H441 
WI-38 
12 
8 
2 
55 
29 
35 
24 
17 
12 
79 
41 
46 
48 
21 
22 
86 
54 
72 
72 
27 
35 
89 
65 
83 
'Cells were infected with AvlLacZ4 at the MOIs indicated and cultured for the times 
indicated and then stained with X-gal. AvlLacZ4 expresses nuclear targeted fl-galactosidase 
thus making scoring of positive cells clear and provides a minimum estimation of the 
efficiency of gene transfer. 
AvlCF2 on cell viability, H441, and A549 cells (both human bronchoalveolar carcinoma cells), 
and human lung WI-38 cells were infected with AvlLacZ4 with 10 and 50 pfu/cell and evaluated 
for cell viability and morphology. Attachment and growth was normal during the period of 
transduction and the extent of transduction was noted to be directly related to MOL Cells 
transduced with AvlLacZ4 at both MOIs demonstrated normal morphology, no alterations 
cellular attachment to the dish and no alterations in viability. As stated earlier, evidence of gene 
transduction was demonstrated by the intense nuclear X-gal staining (Figure 30). 
The potential effect of viral transduction on host cells was further evaluated at the molecular 
level by testing the potential for AE1 adenoviral vectors to alter host protein synthesis. HeLa 
cells were infected with either AvlCFl or AvlCF2 MOI 50 pfu/cell. Cells were cultured at 37° 
and humidified 5% C0 2 atmosphere for 48 hours and labelled with [ 35 S]methionine during the 
last 24 hours of infection. After the labelling period, total cellular proteins were extracted and 
evaluated by SDS-PAGE (Figure 31), as described by Rosenfeld et.al., 1992. As expected, 
wild-type Ad5 adenovirus markedly altered the genetic program of cells as evidenced by a severe 
reduction in the number and quantity of host cell proteins synthesized. In contrast, AvlCFl- 
transduced cells showed a pattern of host protein synthesis identical to that of un-transduced 
cells. Similar AvlCF2 transduced and non-transduced cells also showed an identical pattern of 
protein synthesis. Thus, at the biochemical level, the AvlCF2 vector appears to have no 
significant negative effects on the genetic program of the host cell at the level of general protein 
synthesis. 
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Recombinant DNA Research, Volume 17 
