communication) . The details of the fabrication of AdCFTR have been pub- 
lished (see appendix 1) . The fabrication of AvlCFl is described in Figures 
2.4- A, 2.4-B and 2.4-C. The plasmid pS2CFTR (Figure 2.4-B) was constructed 
by combining the base shuttle plasmid pS2 (Figure 2.4-A) with the entire 
coding sequence of the CFTR cDNA contained within the plasmid pBQ4.7 
(Riordan et al . , 1989; GenBank accession No. M28668; L-C Tsui, personal 
communication) . The final genome of AvlCFl includes (left to right) se- 
quences from pS2CFTR (1-5667 bp in AvlCFl) and sequences from Ad-dl327 
(5668-38,397 bp in AvlCFl). The pS2CFTR derived sequences provide AvlCFl 
with the left ITR, origin of replication, packaging signal (VO , Ela 
promoter/enhancer element, major late promoter and tripartite leader, and 
the CFTR cDNA. The Ad-dl327 derived sequences provide AvlCFl with all of 
the necessary remaining elements (except for the products of the deleted El 
sequences which are provided in trnns by the 293 cells used for production, 
see below) to produce a replication deficient, recombinant infectious 
virus . 
The base shuttle plasmid pS2 was constructed using adenovirus sequences 
derived from Ad-dl327, a mutant adenovirus identical to Ad5 except for the 
minor 2 bp deletion in the VA-I promoter and a deletion of the viral genome 
within the E3 region (sequences 78.5 to 84.7 m.u. are deleted, see Figure 
2.4- C). The Ad-dl327 sequences in pS2 are the left hand end of Ad-dl327 (0- 
1.25 m.u.) including the left hand ITR, origin of replication packaging 
signal (VO and Ela promoter/enhancer. This is followed (left to right) by 
the MLP (16.1 to 16.8 m.u. in Ad-dl327) and the tripartite leader sequences 
from Ad-dl327 (16.8 to 16.9, 19.8 to 19.9 and 26.8 to 27.0 m.u. in Ad- 
dl327). Each of these elements from Ad-dl327 were prepared using PCR ampli- 
fication of Ad-dl327 DNA as a template. The combined elements from Ad-dl327 
were cloned as a Notl-Xbal fragment to a pSKII + derived plasmid containing 
a 2913 bp Bglll - Hindlll fragment of Ad-dl327 (sequences from position 
3328 to 6241 of Ad-dl327, identical to positions 3328 to 6241 of Ad5 (see 
Appendix 3) which had been cloned as a blunt fragment into the Xhol site of 
the pSKII + plasmid (GenBank accession No. 52328). 
The plasmid pBQ4.7 includes a PstI insert of 4.7 kb encompassing the entire 
coding region of the CFTR cDNA and flanking sequences beginning with the 5' 
untranslated sequence at position 72 and ending with the 3' untranslated 
sequence at position 4725 (see GenBank accession No. M28668 for numbering). 
The CFTR cDNA with these flanking sequences were removed from this plasmid 
by cleavage with Spel and Clal and cloned into pS2 (cut with Spel and Clal) 
to form pS2CFTR (Figure 2.4-B). 
For the final construction of AvlCFl, DNA was purified from Ad-dl327 by 
proteinase K digestion, phenol chloroform extraction and dialysis against 
10 mM Tris-Cl , 1 mM EDTA, pH 7.4. The DNA was cleaved with Clal (Figure 
2.4- C), fractionated by agarose gel electrophoresis, and the large (35 kb) 
fragment purified by phenol chloroform extraction and dialysis. The puri- 
fied large Clal fragment of Ad-dl327 (5 /ig) and linear Notl-Kpnl pS2CFTR 
DNA were contransf ected with CaPO*, into 293 cells [50% confluent, 6 cm 
plate] . 
293 cells are used for the production of both AdCFTR and AvlCFl. 293 cells 
Recombinant DNA Research, Volume 16 
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