Checkpoints in Adenoviral Production Figure 3. Detection and effects of E1A in HAEC. (A) DNA was extracted from cesium chloride purified Ad or HEK293 cells and PCR was performed with primers for E1A. Ad 3 shows E1A contamination. (B) VEGF-induced tube formation in HAEC infected with either Ad containing the GOI or Ad coding for the GOI and the contaminant E1A. doi:10.1371/journal.pone.0023160.g003 vector and GOI sequence or, if available, within the affinity tag sequence of the GOI. Primer II binds at the sequence downstream of GOI and ensures amplification of only the Ad sequence. The PCR products were checked for specific amplification and size via agarose gel separation, and DNA sequencing services were used to determine the authenticity of the PCR product (Figure 2B). Depending on the size of GOI, one or more sequencing primers (Primer III) should be designed to cover the entire sequence. When using multiple primers, contiguous primers should be designed to generate overlapping sequences. By using this method we identified a contaminant during an attempt to create an Ad. In this particular case we found 3 different populations of Ad from the initial viral plaque. In Figure 2C, the chromatograms show 1) the desired mutation C118, 2) a mixed virus population of C118 contaminated with S118, and 3) a completely contaminated clone with S118. With this PCR-based method it is not only possible to detect cross-contamination but also accidental mutation that can happen while the virus is amplified from a single copy to up to 1012 viral particles per ml. This screening technique ensures that only viral clones with the correct DNA sequence are propagated and provides assurance that results obtained with Ad can be accurately interpreted. Following amplification in HEK293 cells, high titer virus can be purified using the cesium chloride density centrifugation method, which yields high purity viral stock. We propose References 1. Nevins JR (1986) Control of cellular and viral transcription during adenovirus infection. CRC Crit Rev Biochem 19: 307–322. 2. Wu L, Rosser DS, Schmidt MC, Berk A (1987) A TATA box implicated in E1A transcriptional activation of a simple adenovirus 2 promoter. Nature 326: 512–515. 3. Schaack J, Bennett ML, Colbert JD, Torres AV, Clayton GH, et al. (2004) E1A and E1B proteins inhibit inflammation induced by adenovirus. Proc Natl Acad Sci U S A 101: 3124–3129. 4. Ginsberg HS, Lundholm-Beauchamp U, Horswood RL, Pernis B, Wold WS, et al. (1989) Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proc Natl Acad Sci U S A 86: 3823–3827. 5. Lochmuller H, Jani A, Huard J, Prescott S, Simoneau M, et al. (1994) Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (delta E1 + delta E3) during multiple passages in 293 cells. Hum Gene Ther 5: 1485–1491. routine screening of this stock for the presence of contaminating replication competent Ad. Traditional sequencing protocols for the viral genome rely on reverse transcription of mRNA produced in Ad transfected cells; however, using proteinase K extraction, we purified viral DNA and directly tested for E1A using PCR. We tested four viruses amplified in HEK293 cells and found E1A contamination present in one Ad stock (Figure 3A). Using the contaminated Ad and an Ad containing the same GOI but no E1A contamination, we infected human aortic endothelial cells (HAEC) and measured cellular migration using a tube formation assay in Matrigel-coated plates. Ad contaminated with E1A showed a marked increase in tube formation over the same virus without E1A (Figure 3B) in response to VEGF, demonstrating the possible confounding impact of E1A contamination in cell culture studies. Testing for E1A contamination after purification of Ad should be standard practice in order to ensure the integrity of results in Ad- transduced cells. Author Contributions Conceived and designed the experiments: DH AE JB RAC MB DP. Performed the experiments: DH AE JB. Analyzed the data: DH AE JB MB DP. Contributed reagents/materials/analysis tools: DP. Wrote the paper: DH AE JB RAC MB DP. 6. Luo J, Deng ZL, Luo X, Tang N, Song WX, et al. (2007) A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc 2: 1236–1247. 7. Adachi T, Pimentel DR, Heibeck T, Hou X, Lee YJ, et al. (2004) S- glutathiolation of Ras mediates redox-sensitive signaling by angiotensin II in vascular smooth muscle cells. J Biol Chem 279: 29857–29862. 8. Lavine JA, Raess PW, Davis DB, Rabaglia ME, Presley BK, et al. (2010) Contamination with E1A-positive wild-type adenovirus accounts for species- specific stimulation of islet cell proliferation by CCK: a cautionary note. Mol Endocrinol 24: 464–467. 9. Kuwano K, Nomoto Y, Kunitake R, Hagimoto N, Matsuba T, et al. (1997) Detection of adenovirus E1A DNA in pulmonary fibrosis using nested polymerase chain reaction. Eur Respir J 10: 1445–1449. PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23160