Checkpoints in Adenoviral Production Figure 1. Homologous recombination of HEK293 Ad5 genomic DNA with the Ad construct. HEK293 cells contain bases 1–4345 of Human Ad serotype 5 genome, including the packaging signal (y) and E1 region. Overlapping Ad5 sequences in 2nd generation Ad constructs can undergo single or double homologous recombination to yield replication competent Ad. doi:10.1371/journal.pone.0023160.g001 the early viral genes and how to monitor the authenticity of Ad during the amplification process via a simple PCR method. Materials and Methods Ad constructs were prepared and cesium chloride purification was performed according to the previously reported methods [6,7]. Isolation of Ad DNA and PCR for E1A DNA was isolated from cesium chloride-purified Ad using proteinase K extraction with the RedExtract-N-Amp Tissue PCR kit (Sigma). Isolated Ad DNA was tested for the presence of E1A contamination using the following primer sets as reported by Lavine et al. and Kuwano et al. [8,9]: E1A nested outer primers: 59-CTGCCACGGAGGTGTTAT- TACC-39 and 59-CTCAGGTTCAGACACAGGACCT-39 E1A nested inner primers: 59-GAACCACCTACCCTTCAC- GAACTG-39 and 59-GTGGCAGGTAAGATCGATCACCTC-39. E1A primer set: 59-TATGCCAAACCTTGTACCGGAGGT- 39 and 59-CCGGGGTGCTCCACATAATCT-39 Nested PCR reactions were performed sequentially. All reactions were performed using REDExtract-N-Amp PCR Reaction Mix containing JumpStart Taq polymerase (Sigma Aldrich) using an Endurance TC-512 Thermocycler (Techne Inc.). Primers were added at a final concentration of 5 mM to 16 PCR Reaction Mix containing 4 ml DNA for a final volume of 20 ml. Nested PCR was performed with initial denaturation at 94 degrees for 3 mins followed by 30 cycles with denaturation for 1 min at 94 degrees, annealing for 45 secs at 63 degrees and extension for 1 min at 72 degrees. Final extension was held for 10 mins at 72 degrees. Confirmation of PCR products was performed using the E1A primer set and was performed with initial denaturation at 94 degrees for 3 mins followed by 30 cycles with denaturation for 1 min at 94 degrees, annealing for 30 secs at 60 degrees and extension for 45 secs at 72 degrees. PCR products were analyzed on a 1% agarose gel containing 200 ng/ml ethidium bromide (Bio-Rad) in 16 TAE buffer. Bands were visualized using the Bio-Rad Gel-Doc system. Isolation of Ad DNA and PCR for GOI DNA was isolated from early amplifications by taking a small portion of the cell suspension before freeze/thawing, obtaining a cell pellet by centrifugation (5 min, 1000 rcf) and performing proteinase K extraction with the RedExtract-N-Amp Tissue PCR kit (Sigma). Primer I (within affinity tag): 59-CAAATGGGTCGG- GATCTGTA-39 Primer I (within pshuttle-CMV): 59- ACCGTCAGATCCGC- TAGAGA- 39 Primer II (downstream GOI): 59-GATCCGGTGGATCGGA- TAAA-39 Primer III (Sequencing Primer): 59-GATCCAGAAC- CATTTTGTG-39 Primers were added at a final concentration of 0.5 mM to 16 PCR Reaction Mix containing 3–7 ml DNA (depending on viral titer). PCR reaction was performed with an initial denaturation at 94 degrees for 3 mins and annealing at 60 degrees for 3 min, followed by 30 cycles with denaturation for 1 min at 94 degrees, annealing for 30 secs at 60 degrees and extension for 30 secs at 72 degrees. Final extension was held for 10 mins at 72 degrees. 5 ml of PCR product was analyzed for size as described above. The remaining PCR reaction was processed with a PCR cleanup kit (Qiagen) and submitted for sequencing using the Tufts sequencing core facility. Results and Discussion To specifically amplify only GOI and not the HEK cell endogenous gene, we designed a set of PCR primers (Figure 2A), where Primer I binds either at the junction between the upstream Figure 2. Contamination of Ad constructs during replication. (A) Primer locations for PCR of Ad and subsequent sequencing. Primer I and primer III ensure PCR of viral DNA by overlapping either with an affinity tag or part of the construct sequence. (B) Procedure used for sequencing Ad. (C) Sequencing results from early stage Ad amplification demonstrating contamination of the GOI. doi:10.1371/journal.pone.0023160.g002 PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23160