Poxvirus Vector Development Figure 5. Cre-mediated recombination of vaccinia virus DNA is dependent upon loxP site orientation. (A) PCR analysis of the I4L locus using primers flanking the yfp-gpt cassette of two purified DI4L strains generated from either the pDGloxPKODEL-DI4L or pDGloxPKOINV-DI4L vectors before (BSC-40) and after (U2OS-Cre) Cre-recombination. (B) PCR analysis of two purified DI4L strains generated from the pDGloxPKOINV-DI4L vector with primers amplifying inside and outside the yfp-gpt cassette before (BSC-40) and after (U20S-Cre) Cre-recombination. (C) Western blot analysis of two purified DI4L strains generated from either the pDGloxPKODEL-DI4L or pDGloxPKOINV-DI4L vectors for I4, YFP, I3 (positive control for infection) and actin before (BSC-40) and after (U20S-Cre) Cre-recombination. doi:10.1371/journal.pone.0024643.g005 were compared to ECTV-wt, ECTV-DEVM002-yfp-gpt, and ECTV-DEVM002’ infected cells for YFP fluorescence. Only those cells that were infected with recombinant ECTV-DEVM002-yfp-gpt expressed detectable levels of YFP fluorescence. Discussion Poxviruses have been, and will continue to be, important therapeutics for the prevention and treatment of human diseases [2,3,10,11,12,13,19]. Arguably, the application of VV for the eradication of smallpox has been one of the most important medical advances in human history. A variety of poxvirus based vaccine vectors have been developed for both human and veterinary infectious diseases and more recently as agents for the treatment of cancer [2,7,8,10,13,14,15,16,17,19,20]. During therapeutic development it is desirable to use marker genes for both construction of novel vectors but also for pre-clinical and even early phase clinical experimentation. Unfortunately as products mature, there becomes a transition point where marker genes are no longer necessary and, in the view of regulatory agencies, may even compromise the safety of the vector [29]. The SEM system provides a faster, simplified and more efficient means to create marker-free recombinant poxviruses [31,32,35]. As outlined in Fig. 1A, the SEM cloning strategy can be summarized in three basic steps: transfection of poxvirus-infected cells with an SEM vector, purification of resulting recombinants through FACS and/or drug selection, and finally removal of the selectable marker by virus passage in Cre-expressing cells. The first generation pSEM-1 vector (Fig. 1B) was created to demonstrate the ‘‘proof of principle’’ that it was possible to create marker-free recombinant poxviruses with our system, while stably retaining a functional transgene (in our case luciferase) during the Cre-recombination reaction (Fig. 2B). The second generation SEM plasmids (Figure S2) broaden the applicability of the SEM system to other poxvirus loci, and other poxvirus genera by the insertion of two multiple cloning sites (MCS) flanking the yfp-gpt cassette and loxP sites. Sequences homologous to PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24643