M.J. Welsh and A.E. Smith, RAC Application 
nostril with the posterior balloon inflated. The viral solution remained in contact with the 
nasal mucosa for 30 minutes. At the end of 30 min, the remaining viral solution was 
removed by suction. The balloons were deflated, the catheters removed, and the monkey 
allowed to recover from anesthesia. Monkey A received the CsCl- purified virus and 
Monkey B received the crude virus. 
To obtain nasal epithelial cells from an anesthetized monkey, we first impregnated the 
nasal mucosa with 5 drops of Afrin (0.05% oxymetazoline hydrochloride, Schering- 
Plough) and 1 ml of 2% Lidocaine for 5 min. We then used a cytobrush (the kind typically 
used for Pap smears) to gently rub the mucosa for about 3 seconds. For tracheal brushings, 
we used a flexible fiberoptic bronchoscope; a 3 mm cytology brush (Bard) was advanced 
through the bronchoscope into the trachea and a small area was brushed for about 3 
seconds. We repeated this procedure twice to obtain a total of ~10^ cells. To obtain 
pharyngeal epithelia, we rubbed a cotton-tipped applicator over the back of the pharynx 2-3 
times. The resulting cells were dislodged from brushes or applicators into 2 ml of sterile 
PBS. 
Results 
To detect vector-generated mRNA we used reverse transcriptase PCR (RT-PCR). We used 
PCR primers in both the adenovirus sequence and the Lac Z sequence. RT-PCR of brushed 
cells revealed a band of the correct size that hybridized with a 6-Gal probe, consistent with 
the presence of 6Gal mRNA in the samples from both monkeys. RT-PCR was positive on 
days 3 and 7 and was also positive with cells from the control nostril of monkey A on day 
3. 
We also confirmed the presence of mRNA by using in situ hybridization to detect 
transcripts in nasal epithelial cells obtained by brushing. At day seven after infection, we 
observed specific hybridization with the antisense, but not the sense, probe in cells from the 
right nostril. Only a rare positive cell was detected from the left (control) nostril. 
Cells obtained by nasal brushing were also examined for 6-galactosidase activity by 
staining with X-Gal. X-Gal stains revealed blue-stained cells with clear nuclear 
localization of the pigment through day 7 post infection. X-Gal stains from the right nostril 
of monkey A, revealed approximately 1% of cells stained with nuclear-localized blue stain; 
these cells were predominantly ciliated respiratory cells. Figure 12 shows an example of 
the ciliated respiratory epithelial cells obtained by brushing (Wright stain) and shows cells 
with blue-stained nuclei. Monkey B had a lower percentage of stained cells from the 
infected nostril and none from the control side. Blue cells were not observed on day 14 or 
in subsequent samples. 
Recombinant DNA Research, Volume 16 
[893] 
