M.J. Welsh and A.E. Smith, RAC Application 
A 
ITR and Replication origin 
E 1 a Enhancer and Packaging Signals 
El a Promoter Region 
El amRNA5' Untranslated leader 
47 bp Synthetic Linker 
CFTR cDNAFragnent4.5 kb 
E 1 b 3' Untranslated Region 
Protein IX Coding Sequence 
Figure 2. Schematic of 5’ 5.6kb of Ad2/CFTR-1 
mRNA. Within the CFTR cDNA there are two differences from the published (5) cDNA 
sequence. An A to C change at position 1990 of the CFTR cDNA (published CFTR cDNA 
coordinates) which was an error in the original published sequence, and a T to C change 
introduced at position 936. The change at position 936 is silent but increases the stability 
of the cDNA when propagated in bacterial plasmids (22,57). The 3' untranslated region 
comprises 6 bp of linker sequences and approximately 485 nucleotides (following removal 
of spliced out nucleotides) derived from the Elb mRNA. Although the adenovirus protein 
IX coding sequence is embedded within the 3' Elb derived untranslated region, the protein 
IX transcript is independent of the CFTR transcript. 
Although the activity of CFTR can be measured readily by electrophysiological methods, it 
is relatively difficult to detect biochemically or immunocytochemically, particularly at low 
levels of expression (22,32). We, therefore, constructed a very similar adenovirus vector 
encoding B-galactosidase containing a nuclear localization signal. This vector is useful for 
some efficacy and marking experiments, and also for safety studies. The Ad2/BGal-l 
vector is identical to the CFTR virus, except that in place of the CFTR cDNA, an 
approximately 3.3 kb sequence encoding E. coli 6-galactosidase fused to an SV40 T- 
antigen nuclear localization signal was inserted (107). 
Because this vector is smaller (approximately 101% of wild-type) than Ad2/CFTR-1, it 
replicates more readily in 293 cells (i.e., 10 8 - 10 9 PFU/ml vs. 10 7 - 10 8 PFU/ml for 
Ad2/CFTR-1). For some safety studies, we used the Ad2/6Gal-l virus as a more sensitive 
means for testing the possibility of vector replication. 
B.l.a.(2) What regulatory elements does the construct contain (e.g.. promoters, enhancers, 
polvadenvlation sites, replication origins, etc.)? From what source are these elements 
derived? Summarize what is currently known about the regulatory character of each 
element. 
All the regulatory elements used in the construct are the endogenous Ad2 control elements. 
The promoter/enhancer responsible for transcription of the CFTR cDNA is the El a 
Recombinant DNA Research, Volume 16 
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