A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses Julia L. Rintoul1,2., Jiahu Wang2., Don B. Gammon 3 , Nicholas J. van Buuren 3 , Kenneth Garson 2 , Karen Jardine2, Michele Barry3, David H. Evans3, John C. Bell1,2* 1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada, 2 Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Canada, 3 Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Canada Abstract Background: Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value. Methodology/Principal Findings: Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene. Conclusion/Significance: The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications. Citation: Rintoul JL, Wang J, Gammon DB, van Buuren NJ, Garson K, et al. (2011) A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses. PLoS ONE 6(9): e24643. doi:10.1371/journal.pone.0024643 Editor: William P. Halford, Southern Illinois University School of Medicine, United States of America Received December 23, 2010; Accepted August 16, 2011; Published September 8, 2011 Copyright: 2011 Rintoul 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 grants from the National Cancer Institute of Canada, Terry Fox Foundation, Natural Sciences Engineering Research Council, and the Canadian Institutes of Health Research to JB, DE and MB. JLR was supported by a CIHR Doctoral award: Frederick Banting and Charles Best Canada Graduate Scholarship. JW was supported by a Lymphoma Research Foundation of Canada Research Fellowship. DBG was supported by a Ralph Steinhauer Award of Distinction from the Province of Alberta. NJVB was supported by a Doctoral NSERC Canada Graduate Scholarship and an Alberta Heritage Foundation for Medical Research Incentive Award. MB holds a Tier I Canada Research Chair, a Howard Hughes International Scholar Award and an Alberta Heritage for Medical Research Senior Scholar award. The funders had no role in the 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: jbell@ohri.ca . These authors contributed equally to this work. Introduction Poxviruses comprise a large family of double-stranded DNA viruses that infect a wide range of hosts. Vaccinia virus (VV) is the prototypic member of the Orthopoxvirus genus and the best-studied virus in the poxvirus family. Since the eradication of smallpox [1], VV and other poxvirus species have continued to be used for the treatment of human disease [2,3] in part because a greater understanding of poxvirus biology has led to safer and more efficacious poxvirus-based therapeutics. The poxvirus genome is easily genetically modified and can accommodate inserts exceed- ing 25 kb [4] using strategies that are dependent upon virus- encoded homologous recombination [5,6]. Using these approach- es, recombinant VV has since proven to be valuable as a vector for gene therapy in a number of therapeutic applications [4,7,8,9,10,11,12,13,14,15]. Similarly, other members of the poxvirus family have also been explored for their potential as viral vectors for therapeutic purposes [9,10,16,17]. Genetically engineered poxviruses that express immunogens from other infectious agents have shown some promise as novel vaccines against diseases like acquired immunodeficiency syndrome [11], malaria [12], tuberculosis [18], and cancer [7,8,10,13]. As a cancer vaccine, poxviruses have the potential to generate a strong anti-tumoural immune response, especially when genetically modified to express cytokines like IL-2 [14] or cell surface receptors like CD70 that are indicative of oncogenic transforma- tion [15]. Lastly, poxviruses have been successfully engineered as oncolytic agents, offering the advantage of a strong anti-tumoural immune response combined with cancer cell-specific replication [7,16,17,19,20]. A number of these poxvirus candidates have advanced to human clinical trials [10,11,12,13,19], highlighting the therapeutic potential of poxvirus recombinants. Poxvirus recombinants are typically produced by constructing a plasmid containing the gene(s) of interest flanked by DNA sequences homologous to the desired target locus, followed by transfection of the plasmid into VV infected cells to allow for PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24643