M.J. Welsh and A.E. Smith, RAC Application 
We also tested for the presence of Ad2/6Gal-l DNA by PCR of the brushed samples. 
Although the results do not reveal whether the virus was transcribing 6-galactosidase 
mRNA or producing 6-galactosidase protein, the test is the most sensitive assay for the 
presence of viral DNA. Moreover, we expect that some of the viral DNA detected by PCR 
will be generating protein below the threshold of detection of the tests employed. We used 
the same primers used for the RT-PCR. After 2 weeks, DNA was detected only from the 
right (experimental) nostril. Viral DNA encoding 6-galactosidase was no longer detected 4 
weeks after virus application. 
Conclusions 
The results of this study showed that a gene can be expressed by a recombinant adenovirus 
in vivo in primate respiratory epithelial cells. In the absence of data on the sensitivity of the 
X-gal staining procedure or on quantitation of the PCR reaction, it is difficult to estimate 
the number of nasal cells that took up the Ad2/6Gal-l vector DNA. We also found the 
transient presence of 6-galactosidase protein and mRNA in the control nostril and in the 
pharynx. We think the virus most likely reached the contralateral nostril and pharynx via 
drainage by gravity after we released the posterior occlusion or it may have been caused by 
the monkey putting its finger from one nostril to another after recovery from anesthesia. 
b. Ad2/CFTR-1 applied to the nasal epithelium. 
Procedure 
For this study, we used 3 monkeys (C, D, & E). As of late September, they had been 
followed for 4 weeks after virus application. 
We applied the Ad2/CFTR-1 construct to the nasal epithelium exactly as described above 
for studies with the Ad2/6Gal-l virus, with the exception that the epithelium was not 
pretreated with dithiothreitol and neuraminidase. In each monkey, we applied 2.5 x 10 9 
PFU in 0.4 ml of CsCl- purified, dialyzed virus to the right nostril. Airway epithelial cells 
were obtained by brushing, as described above. 
Results 
RT-PCR of brushed cells was positive on day 3 and 6 on the right, but not the left nostril. 
After infection RT-PCR (using primers in the adenovirus sequence and theCFTR 
sequence) revealed a band of the correct size which hybridized with a CFTR probe, 
consistent with recombinant CFTR mRNA in the samples. 
Cells were also processed on cytospin slides and prepared for immunocytochemistry using 
monoclonal antibodies to CFTR. On day 3 after infection, positive immunofluorescence 
was detected in all three monkeys, predominantly in cells from the right nostril, although a 
few cells stained positive from the left, control nostril. Figure 13 shows an example. 
CFTR was also detected by immunostaining in two of the monkeys at one week after 
infection. CFTR immunofluorescence was negative in samples from monkeys recei ving 
Ad2/6Gal-l. (Note, that we have not determined whether our antibodies to human CFTR 
crossreact with endogenous monkey CFTR.) 
PCR of CFTR DNA encoded by Ad2/CFTR-1 was positive in cells from the right nostril, 
but not the left nostril or pharynx, for two weeks after infection in all of the monkeys. 
Recombinant DNA Research, Volume 16 
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