variety of indicator cell lines are exposed to 293 supernatants, passaged, and 
inspected for cytopathic effects. 
Detection and quantitation of low level wild type adenovirus in high titer 
preparations of the recombinant Ad5-CB-CFTR virus is confounded by the 
observation that El deleted viruses will cause cytopathic effects (CPE) in non El 
expressing cells, such as HeLa cells infected at high MOI’s. Mechanism(s) for this 
relate to the direct toxic effect of high concentrations of viral proteins such as fiber 
and the possibility of low level viral replication of El deleted virus at high MOI. We 
find it necessary to dilute the concentrated stock at least 100 to 1000 fold to avoid 
CPE in most indicator cell lines. Two types of assays will be performed. A typical 
virus preparation contains 5 x lO 1 ^ particles/ml based on OD at 260 nm and ~ 10^ 
functional virions/ml based on pfu or assays that measure CFTR protein expression 
(immunocytochemistry or SPQ functional analyses). The first assay is based on the 
ability of wild type Ad (but not El deleted Ad) to replicate in non-El expressing cell 
lines. An aliquot of each preparation representing 5% of the total will be diluted 
and exposed to a variety of indicator cell lines. The cells are passaged for 2 weeks 
to allow spread of wild type virus and subsequently examined for CPE. Specifically, 
for each ml of stock, 5 /id is removed, diluted and exposed to 10 x 15 cm plates of 
confluent indicator cells. Preliminary results with purified preps of recombinant 
viruses have failed to demonstrate CPE under these conditions. The sensitivity of 
the assay is currently being defined in reconstitution experiments. 
Another assay has been developed to detect and quantify wild type Ad5 in 
the stocks of recombinant virus. The potential sources of wild type Ad5 are 
contamination from the initial transfection or recombination with Ad5 sequences 
originally transfected into the 293 cells. The assay is based on PCR detection of El 
sequences in DNA isolated from the stock of recombinant virus. Figure 25 shows an 
agarose gel of PCR products in which 125 ng of total cellular DNA was 
supplemented with varying amounts of wild type Ad viral DNA (from 100,000 to 0.1 
copies of Ad5 per reaction). Amplification for 35 cycles afforded a sensitivity equal 
to 100 viral molecules/reaction (panel A). However, when the initial reaction 
products were used as a template for another 35 cycles of amplification using nested 
primers, the sensitivity was improved to 1 viral molecule/reaction (panel B). DNA 
will be isolated from a five percent of each viral stock and subjected to the PCR 
analysis. Five percent of a 2 ml viral stock should yield 2-20 mg of DNA which can 
be analyzed in 4 to 40 reaction tubes. 
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Recombinant DNA Research, Volume 17 
