Checkpoints in Adenoviral Production: Cross-Contamination and E1A Dagmar J. Haeussler1., Alicia M. Evangelista1., Joseph R. Burgoyne1, Richard A. Cohen1, Markus M. Bachschmid1*", David R. Pimental2" 1 Vascular Biology Section, Boston University Medical Center, Boston, Massachusetts, United States of America, 2 Myocardial Biology Unit, Boston University Medical Center, Boston, Massachusetts, United States of America Abstract Adenoviruses are widely used for overexpressing proteins in primary mammalian cells. Incorporation of the early viral gene, E1A, or viral cross-contamination can occur during amplification, and identification of these products is crucial as the transcription of unwanted genetic material can impact cell function and compromise data interpretation. Here we report methods for evaluation of contaminating adenovirus and E1 viral DNA. Citation: Haeussler DJ, Evangelista AM, Burgoyne JR, Cohen RA, Bachschmid MM, et al. (2011) Checkpoints in Adenoviral Production: Cross-Contamination and E1A. PLoS ONE 6(8): e23160. doi:10.1371/journal.pone.0023160 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received May 31, 2011; Accepted July 7, 2011; Published August 3, 2011 Copyright: 2011 Haeussler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health (HL031607-28 to R.A.C., PO1 HL 068758 and R37 HL104017 to D.J.H., J.R.B., and M.M.B., and HL007501-26 to A.E., and K08HL071563 to D.P.) and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the awarding offices. This work was also supported by a Sir Henry Wellcome postdoctoral fellowship from The Wellcome Trust (Sponsor Reference 085483/Z/ 08/Z; to J.R.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bach@bu.edu . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction The generation and use of adenoviruses (Ad) in physiological research has become a routine procedure due to their ability to increase protein expression in primary mammalian cells that cannot be efficiently transfected with plasmid DNA. Creating and amplifying an Ad is a time and labor intensive process; therefore it is necessary to determine as early as possible that the gene of interest (GOI) is free of mutations and is not contaminated with another coding sequence. This is particularly pertinent when creating more than one Ad at a time using site directed mutagenesis (i.e. wild type and mutant), as it is not possible to verify the authenticity of the final adenovirally expressed protein using standard immunoblot techniques. Although it is common practice to screen the Ad cosmid DNA for errors, in our experience the stage at which Ad are most susceptible to unwanted contamination, and therefore the most important to screen, is during the initial formation of a viral plaque. During this period of time a single contaminating viral particle, i.e. from aerosolization of another Ad used in the same sterile hood, or an Ad that is amplified in the same incubator as the single clones, is enough to cause contamination, which may replace expression of the intended GOI with that of the contaminating virus. We therefore propose routine evaluation of the initial viral plaques should be done using a simple PCR- based method and suggest routine decontamination of incuba- tors used to amplify Ad. Refined vector systems, such as AdEasy, make Ad production a relatively straight forward procedure. This system is based on the human Ad serotype 5 genome and belongs to the so called 2nd generation Ad constructs that omit the early viral genes, E1 and E3, but maintain the viral packaging signal and inverted terminal repeat (ITR) required for viral transcription. The benefits of using 2nd generation Ad are that they can accommodate a GOI up to 7.5 kb. Using a bacterial plasmid containing left and right arm flanking homology domains, the GOI is inserted into the site of the disrupted E1A by homologous recombination. E1A is a viral transcription factor required for induction of early genes necessary for viral replication, including E1B and E2 [1,2], progression of the cell cycle, and inhibition of apoptosis [2]. E3, though not technically required for viral replication, plays an important role in suppression of host immunity [3,4]. In the absence of E1A and E3, the recombinant Ad can infect but not replicate in most cells. Since 2nd generation Ad lack E1 and E3, rendering them non- replicative, they must be amplified in packaging cell lines that express E1 gene products. In HEK293 cells, which are transformed with bases 1–4355 (containing full-length E1) of sheared human Ad5 genome, the Ad with GOI insert can be packaged and amplified via E1A and E3 supplied in trans. However, because many vector systems maintain significant portions of the human Ad5 genome, single or double homologous recombination events between the transformed HEK293 cells and the viral cosmid can occur, resulting in the production of replication competent Ad contamination (Figure 1). Though rare in early passages of adenovirus, the probability of replication competent Ad contamination increases with large-scale amplifica- tion [5], a phenomenon that has led to the decline of HEK293 cells in adenoviral production for clinical usage but has not translated to the basic science laboratory. Even optimized protocols fail to warn about the possibility of recombination with PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23160