Complementary oligonucleotides encompassing the 3' end of the left-hand ITR and 5' end of 
the CFTR cDNA were designed to "bridge" this region and allow for removal of the p5 
promoter region. Oligonucleotide sequences CFEAV-F and CFEAV-R are shown below: 
5 ' -GGCCACTCCATCACTAGGGGTTCCTCTCGAGC 
3 ' -TGAGGTAGTGATCCCCAAGGAGAGCTCGGGCT 
A 339 bp Aat I3/Eae I fragment containing the 5' end of the left-hand ITR was isolated from 
pUCAX. pAVX was digested with Aat II and Ava I and a three-way ligation performed with 
annealed oligonucleotide, Aat II/Eae I fragment, and pAVX backbone to yield pDCF, 
containing an ITR-CFTR junction without the AAV p5 promoter. 
Correction of the single nucleotide change in the left-hand ITR is diagrammed in figure 7.1.6. 
Complementary oligonucleotides encompassing the 5' end of the left-hand ITR were 
synthesized. Oligonucleotide sequences CFAAB-F and CFAAB-R are shown below: 
5 ' -CAGATCTTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCC 
3 ' -TGCAGTCTAGAAACCGGTGAGGGAGAGACGCGCGAGCGAGCGAGTGACTCCGGC 
A 630 bp Bgl I/Xba I fragment containing the 3' end of the left-hand ITR was isolated from 
pDCF. pUC19 was digested with Aat II and Xba I, and a three-way ligation performed with 
annealed oligonucleotide, Bgl I/Xba I fragment, and the pUC19 backbone to yield pi5CF, 
containing the corrected left-hand ITR and ITR-CFTR junction. 
Correction of the 8 base deletion in the right-hand ITR was performed in two steps (figures 
7.1 .7 and 7. 1 .8). A 632 bp Nde I/Sac I fragment encompassing a portion of the 
polyadenylation sequence and the right-hand ITR was isolated and subcloned into pUC19 
digested with Nde I and Sac I to yield pUCNS. Complementary oligonucleotides 
encompassing the 3' end of the right-hand ITR were synthesized. Oligonucleotide sequences 
CFBN-F and CFBN-R are shown below: 
5 ' -CGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCTTAGATCTCA 
3 ' -CCCGCCGGAGTCACTCGCTCGCTCGCGCGTCTCTCCCTCACCGGAATCTAGAGTAT 
A 180 bp Sac I/Bgl I fragment containing the polyadenylation sequence and the 5* end of the 
right-hand ITR was isolated from pUCNS. p5CF was digested with Sac I and Nde I, and a 
three-way ligation performed with annealed oligonucleotide, Sac I/Bgl I fragment, and the 
p5CF backbone to yield pi3CF, containing the corrected right-hand ITR. 
The final vector containing all changes was constructed as diagrammed in figure 7.1.9. A 680 
bp Aat II/Xba I fragment containing the left-hand ITR and 5' end of the CFTR cDNA was 
isolated from pi5CF. pi3CF was digested with Aat II and Xba I, and the -6200 bp backbone 
fragment containing the remainder of the CFTR cDNA, polyadenylation sequence, and right- 
hand ITR isolated. These fragments were ligated to produce the final plasmid, tgAAVCFpBR. 
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