3.1 In Vitro Evaluation of AvlCF2-Derived Expression of hCFTR mRNA: Human kidney 
epithelial cells 293 cells (Graham 1977) were infected AvlCF2; an appropriate 5.2 kb mRNA 
was detected by northern blot analysis (Figure 16). Likewise, murine respiratory epithelial cells, 
immortalized by our laboratory from SV40 transformed lung cells, were infected with AvlCF2 
at an MOI of approximately 50 pfu/cell. After 48 hours, the RNA was isolated from control 
and infected cells and subjected to S, protection assay designed to detect the endogenous murine 
and transferred human CFTR mRNA’s (Figure 17). The appropriate protected fragments were 
identified by autoradiography demonstrating the synthesis of the human CFTR mRNA in these 
cells at levels greatly exceeding that of the endogenous murine CFTR mRNA. 
3.2 In Vitro Expression of the CFTR protein by AvlCF2: HeLa cells, lacking readily 
detectable CFTR protein by immuno-precipitation assays, were infected with AvlCF2. 35 S- 
Methionine labeled cellular proteins were immuno-precipitated with rabbit anti-hCFTR peptide 
antibody resulting in the identification of a 155 kilodalton protein (Figure 18) migrating as a 
band consistent with the size of the hCFTR polypeptide as previously demonstrated by Chang 
et.al., (1990). Immunocytochemistry was utilized to detect hCFTR with monoclonal CFTR 
antibody (Genzyme, Inc.) after AvlCF2 infection of 293 cells, demonstrating immunostaining 
of the majority of cells (Figure 19). 
3.3 In Vitro Evaluation of AvlCF2 Vector Expression of Functional cAMP Regulated Cl' 
Conductance: Biological function of the AvlCF2 directed CFTR protein has been demonstrated 
by three distinct assays using CFPAC-1 cells. CFPAC-1 cells are pancreatic adenocarcinoma 
cells that were derived from a patient who is homozygote for A508 mutation. CFPAC-1 cells 
fail to respond to cAMP. We have utilized 1) epithelial conductance, 2) SPQ fluorescence and 
3) Cl' 36 efflux studies, to demonstrate in vitro that the CFTR polypeptide directed by AvlCF2 
functions in appropriate manner to alter ion transport. 
Transepithelial Cl' Conductance: CFPAC cells were grown to confluence on support 
matrix infected with AvlCF2, and placed in a Ussing chamber (Figure 20). The conductance 
was determined over time ii^ the presence and absence of forskolin, ATP, carbachol or 
ionomycin. Control CFPAC-1 cells failed to respond to forskolin but had detectable responses 
to carbachol, ATP and ionomycin. In contrast, a marked Cl* conductance was observed in cells 
after infection with AvlCF2 virus at 10 MOI. 
Cl' 36 Efflux: Studies were performed on CFPAC-1 cells before and after infection with 
AvlCF2 virus at 200 MOI. Forskolin (13 /iM) had no effect on Cl" efflux in control, AvlLacZ4 
cells, but stimulated Cl' efflux in the AvlCF2 infected cells (Figure 21). 
SPQ Assay: SPQ is a Cl' sensitive fluorescent dye that once loaded into cells, provides an 
indirect measurement of the intracellular Cl'. CFPAC-1 cells were infected with AvlCF2 virus 
and SPQ fluorescence determined over time in the cell suspensions. Control CFPAC-1 cells 
were not responsive to forskolin, however, after infection with AvlCF2 (10 MOI), a brisk 
decrease in intracellular Cl' was observed, consistent with the efflux mediated by the transferred 
hCFTR (Figure 22). 
3.4 In Vitro Transfer of hCFTR mRNA to Airway Epithelium of Cotton Rats: Cotton rats 
were treated with 3 x 10 8 pfu of AvlCF2 intranasally in 100 /xl sterile media. After 72 hours, 
animals were sacrificed, the lungs sectioned and in situ hybridization performed to identify and 
compare hCFTR mRNA with the endogenous cotton rat CFTR mRNA (Figure 23). hCFTR 
mRNA was readily detected in tracheal, bronchial and occassional alveolar cells using 35 S- 
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Recombinant DNA Research, Volume 17 
