the parent vector, called pgagBA. The human CFTR coding sequences were 
derived from a 4.6 kb SacI fragment which contains the complete coding sequence 
and small 5’ and 3’ untranslated regions (7). The SacI sites were converted to Bell 
sites and the modified fragment was cloned into the BamHI site of pgagBA. This 
vector is called pgagBA-CFTR. An enhancer was introduced into the Xhol site of 
this vector. These sequences were derived from an area 5’ to the immediate early 
(IE) gene of human cytomegalovirus [from Spel (at -580 of IE gene) to PstI (site in 
vector sequence) of CDM1 (76) were subcloned into PUC19. A portion containing 
IE enhancer sequences was removed on a Xhol (from polylinker) to Ncol (-220 of 
the IE gene) fragment [for numbering of IE enhancer see (72)]. Synthetic linkers 
were used to convert the Ncol site to a Xho I site and the modified fragment was 
cloned into the unique Xhol site of pgagBACFTR located 5’ to the b-actin 
promoter. This new vector is called pCMV-BA-CFTR. 
The retroviral vector pCMV-BA-CFTR was digested with Xho I and Nhe I to 
release a fragment (BA-O'I K) containing the chick b-actin promoter, the 4.6 kb 
human CFTR cDNA and a small portion of retrovirus-specific sequences. The 
plasmid pAd5-CMV-lacZ was cut with Sna BI and Not I to remove CMV promoter 
and lacZ structural gene, and the plasmid backbone retaining the CMV enhancer 
and the SV40 polyadenlyation signal was blunted and ligated with the blunted BA- 
CFTR fragment to form the plasmid pAd5-CB-CFTR. For a schematic 
representation of this please see Panel C (Appendix F). 
III.B. Generation of Recombinant Ad5-CB-CFTR Virus 
The recombinant CFTR adenovirus was constructed from the plasmid pAd5- 
CB-CFTR and a modified adenovirus type 5 (Ad5), dl7001, in which the majority of 
the E3 region (map units 78.4-86) was deleted. 
dl7001 was kindly provided by Dr. William Wold from St. Louis. A 
description of this viral genome as provide to us is presented below verbatim. 
dl7001 is derived from rec700, an Ad5-Ad2-Ad5 recombinant which has the Ad5 
EcoRI-A (map unit 0-76), Ad2 EcoRI-D (mu 76-83), and Ad5 EcoRI-B (mu 83-100) 
fragments. dl7001 retains Ad2 sequences from map position 76 to 78.4, and it is 
deleted of sequences from 78.4 to about 86. In terms of the nucleotide numbering 
for the E3 transcription unit dl7001 retains Ad2 sequence from nt -236 to 362 (77). 
The deletion extends from nt 362 in the Ad2 sequence to nt 3382 in the Ad5 
sequence (nt 2437 in the Ad2 sequence corresponds to nt 2482 in the Ad5 
sequence). The total deletion is 2,975 bp. Because of this deletion there are no 
authentic E3 AUG’s or polyA sites. 
pAd5-CB-CFTR was linearized by Nhe I cleavage and cotransfected with the 
large fragment of Cla I-cut dl7001 DNA into 293 cells (a human kidney cell line 
containing a functional E1A gene that provides a trans-acting Ela protein) to allow 
homologous recombination to occur, followed by replication and encapsidation of 
recombinant adenoviral DNA into infectious virions and formation of plaques. 
Individual plaques were isolated and amplified in 293 cells, viral DNA was isolated, 
and recombinant adenoviral plaques containing the human CFTR cDNA were 
identified by restriction cleavage and Southern blot analysis. One of the positive 
plaques was plaque-purified a second time, and designated Ad5-CB-CFTR. This 
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Recombinant DNA Research, Volume 17 
