Second, the ITR, packaging signal (\p), rous sarcoma promoter, the adenoviral tripartite 
leader (TPL) sequence and linking sequences were assembled as a block using PCR amplification 
(Figure 7). The ITR and \J/ (sequences 1 - 392 of Ad-d]327 [identical to sequences from Ad5, 
Genbank accession # M73260]) were amplified (amplification 1) together from Ad-dJ327 using 
primers containing Notl or AscI restriction sites. The Rous sarcoma virus LTR promoter was 
amplified (amplification 2) from the plasmid pRC/RSV (sequences 209 to 605; Invitrogen, San 
Diego, CA) using primers containing an AscI site and a Sfil site. DNA products from 
amplifications 1 and 2 were joined using the "overlap" PCR (amplification 3) with only the Notl 
primer and the Sfil primer. Complementarity between the AscI containing end of each initial 
DNA amplification product from reactions 1 and 2 allowed joining of these two pieces during 
amplification. Next the TPL was amplified (amplification 4) (sequences 6049 to 9730 of Ad- 
dj327 [identical to similar sequences from Ad5, Genbank accession #M73260]) from cDNA 
made from mRNA isolated from 293 cells infected for 16 hr with Ad-d]327 using primers 
containing Sfil and Xbal sites respectively. DNA fragments from amplification reactions 3 and 
4 were then joined using PCR (amplification 5) with the Notl and Xbal primers thus creating 
the complete gene block. 
Third, the ITR-i/'-TPL fragment was then purified, cleaved with Notl and Xbal and inserted 
into the Notl, Xbal cleaved pHR plasmid. This plasmid was designated pAvS6A' and the 
orientation was such that the Notl site of the fragment was next to the T7 RNA polymerase site 
(Figure 8). 
Fourth, the SV40 early poly A signal was removed from SV40 DNA as a Hpal-BamHI 
fragment, treated with T4 DNA polymerase and inserted into the Sail site of the plasmid 
pAvS6A' to create pAvS6 (Figure 8). 
The complete nucleotide sequence of the viral and linker elements within pAvS6 has been 
determined (not shown, but identical to corresponding sequences in pAvS6-CFTR [see appendix 
7]). 
2.3.2 Construction of pAvS6-CFTR: The cDNA encoding the complete polypeptide of the 
human cystic fibrosis transmembrane conductance regulator gene (Genbank accession ft M28668) 
was removed from the plasmid pBQ4.7 (Figure 9; kindly provided by L-C Tsui, Hospital for 
Sick Children, Toronto, Canada) as a Pstl fragment, treated with T4 DNA polymerase and 
inserted into the EcoRV site of pAvS6 (Figure 9). The orientation of this fragment was such 
that the coding sequence of the CFTR cDNA had its 5’ end nearest the T7 RNA polymerase 
promoter site and its 3’ end nearest the T3 RNA polymerase site of the pskll' backbone (not 
shown). The complete nucleotide sequence of the viral elements, linker sequences and the 
human CFTR cDNA were determined by automated sequencing (see appendix 7). 
2.3.3 Construction of Infectious AvlCF2 Virus: Infectious AvlCF2 recombinant adenovirus 
was assembled by co-transfection of linearized pAvS6-CFTR plasmid DNA and the large 
(approximately 35 kb) Clal restriction fragment of Ad-d]327 genomic DNA in 293 cells (a 
human kidney cell line containing 11% of the left end of Ad5 Graham et.al., 1977) cells by 
homologous recombination. To accomplish this, first, the plasmid pAvS6 was linearized by 
cleavage with KpnI. Then viral genomic DNA was isolated from Ad-d]327 virions by extraction 
of viral DNA (Hirt, 1967), cleaved with Clal and the large fragment was isolated by gel 
electrophoresis. Two micrograms of linearized plasmid DNA and adenoviral DNA were then 
mixed and cotransfected into 293 cells by the calcium phosphate method (Figure 10). After 16 
hr, media containing 1 % agarose was overlaid and the cells were incubated in a humidified, 5% 
[372] 
Recombinant DNA Research, Volume 17 
