Figure 3.1.1-C. Evaluation of Ad-CFTR specific CFTR transcripts in human 
respiratory epithelial cells from an individual with CF following in vitro 
infection with Ad-CFTR. Human respiratory epithelial cells were obtained 
during bronchoscopy by cytologic brush, infected with Ad-CFTR and cultured 
as previously described (Appendix 1; Rosenfeld et al . , 1992). RNA was 
extracted, treated with DNase, converted to cDNA, and amplified with a 
primer pair consisting of an adenoviral-specific sense primer and a human 
CFTR-specif ic antisense primer as previously described (Appendix 1) . Each 
DNase - treated sample was used as a PCR template in parallel without conver- 
sion to cDNA to eliminate the possibility of amplifying potentially contam- 
inating viral DNA. PCR products were evaluated by agarose gel electrophore- 
sis followed by Southern hybridization using a nested 32 P-labeled human 
CFTR cDNA probe. Panel A shows the strategy for amplification and hybrid- 
ization. Note that the location of the 5' primer used for amplification of 
the cDNA is located in a region of Ad-CFTR that is virus-specific (as 
compared to CFTR-specif ic) i.e., the transcripts could only be derived from 
Ad- CFTR- directed mRNA, not endogenous CFTR mRNA. Panel B shows data from 
RNA extracted from CF respiratory epithelial cells 24 hr after infection. 
Lane 1 - uninfected cells, without reverse transcriptase (RT) ; lane 2 - 
same as lane 1, with RT; lane 3 - cells after infection with the control 
virus Ad-alAT amplified without RT; lane 4 - same as lane 3, with RT ; lane 
5 - after infection with Ad-CFTR, without RT; and lane 6 - same as lane 5, 
with RT. 
A. 
Ad-CFTR 
Viral . 
sequences J ^^ATG 
MLP [’ 
\f‘ 
Human CFTR sequences 
Viral 
TAG -v. sequences 
Ad-CFTR transcript 5' 
•l 2 3 4 5 6a 6b 7 8 9 10 16 17a 17b 18 19 2021 22 23 24 1 
I I 
Nested probe 
Ad-CFTR transcripts 
B. 
+ + 
Uninfected Ad-alAT Ad-CFTR 
RT 
+ - + 
bp 
809 9 
1 2 3 4 5 6 
Recombinant DNA Research, Volume 16 
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