Appendix 3 
GENERATION OF ACN53 
The ACN53 construct was derived from plasmid constructs and adenoviral DNA in the following manner. 
Plasmid pAd/CMV/p53/Elb" was derived by inserting the p53 cDNA into the plasmid pAd/CMV. 
pAd/CMV/p53/Elb' is based on the pBR322 derivative pML2 and contains, in order, the adenovirus type 
5 inverted terminal repeat (HR), the human cytomegalovirus immediate early promoter-enhancer (CMVp), 
the adenovirus type 2 tripartite leader (TPL), a 1.4 kb cDNA encoding wild-type p53 and adenovirus type 
5 sequences corresponding to Ad5 base pairs 3328 to 5792. To delete the protein EX (pIX) coding 
sequence, pAd/CMV/p53/Elb‘ was cleaved with restriction endonucleases NgoMI and Eel 1 3611 and a 4.6 
kb fragment containing the pML2 backbone, the ITR, the CMVp, the TPL and the p53 cDNA was isolated 
and ligated to a 6.4 Kb Dra I/NgoM I fragment of Ad2 DNA corresponding to Ad2 coordinates 4021- 
10457. This ligation was used to transform El coli DH5oc cells to ampicillin resistance to select for the 
recombinant plasmid. In the resulting plasmid, pAd/CMV/p53/pEX', the pEX coding sequence is not 
present and p53 cDNA is placed adjacent to the polyadenylation signal shared by pIX and Elb. Due to 
the high degree of nucleotide sequence identity of Ad2 and Ad5 in the regions contained in the plasmid 
(>99%) pAd/CMV/p53/pEX" can readily be recombined with Ad5 serotype viruses to yield Ad2/5 genetic 
hybrids which are immunologically serotype 5. To produce ACN53, pAd/CMV/p53/pIX" was cleaved 
with EcoR I to linearize the plasmid and co-transfected into the human embryonic kidney cell line 293 
with Cla I digested Ad5/dl327. Ad5/dl327 is a serotype 5 derivative containing a 1.9 kb deletion in Early 
Region 3 resulting from the deletion of the Xba I restriction fragment extending from Ad5 coordinates 
28593 to 30470. Virus resulting from recombination between the plasmid and viral genomes was 
expanded on 293 cells and analyzed by restriction analysis and p53 western blotting. The resulting virus, 
ACN53, was obtained by plaque purification and further expanded with 293 cells for virus purification. 
Note: The following page contains a diagram of the construction of ACN53. 
Figure 1 . Diagram of the construction of ACN53. The p53 cDNA was first inserted into the plasmid 
pAd/CMV/p53/Elb". In order to delete the pIX gene, this plasmid was modified by removing the adenoviral 
sequences adjacent to the 3' end of the p53 cDNA, replacing them with a fragment of Ad2 genomic DNA extending 
from base pair 4021 to 10457 (Ad2 nucleotide coordinates). The resulting plasmid, pAd/CMV/p53/pIX' was co- 
transfected into 293 cells along with the large fragment of Cla I digested Ad5/dl327. The virus resulting from 
recombination of the plasmid and viral DNA in the 293 cell line was designated as ACN53. ACN53 is deleted for 
adenoviral sequences between nucleotides 357 and 4021 in the El region. In place of this deletion an expression 
cassette was inserted containing the human cytomegalovirus immediate early promoter-enhancer (CMV), the Ad2 
tripartite leader (TPL) and the p53 cDNA . The virus also contains a 1.9 kb deletion within the early region 3 (E3). 
Recombinant DNA Research, Volume 20 
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