Poxvirus Vector Development Figure 6. Generation of a recombinant ECTV using SEM. (A) Map of pDGloxPKODEL-DEVM002 with labeled open reading frames. (B) Schematic displaying the strategy for knock-out of the EVM002 open reading frame from ECTV. (C) PCR analysis of viral DNA. Primers were used to amplify the regions of pDGloxPKO-DEVM002 homology from wild-type ECTV (ECTV-wt), ECTV-DEVM002-yfp-gpt, and ECTV-DEVM002’. (D) DNA sequence analysis of the ECTV-DEVM002’ virus (post Cre passage). (E) Confocal microscopy was used to detect YFP fluorescence in mock infected BGMK cells or BGMK cells infected with ECTV-wt, ECTV-DEVM002-yfp-gpt, or ECTV-DEVM002’. A 406magnification lens was used to detect differential interference contrast (DIC), and YFP fluorescence. doi:10.1371/journal.pone.0024643.g006 any poxvirus gene can be added to the pDGloxPKO vector, as we have demonstrated with the ectromelia virus recombinant construct- ed from pDGloxPKODEL-DEVM002 (Fig. 6). The most labour-intensive part of traditional poxvirus cloning strategies is the isolation and purification of desired recombinants from a heterogeneous population. To simplify the isolation of pure recombinant poxvirus clones, we have included both drug (gpt) and optical (yfp) selection strategies in the SEM system. The flexibility and simplicity of this approach ensures that the SEM system will be amenable to any research laboratory. MPA drug selection is relatively inexpensive and historically has shown to be an efficient means to purify poxvirus recombinants [25]. Indeed, we have shown here that MPA selection alone is sufficient to isolate a pure recombinant poxvirus population in as few as three passages (e.g. VV-DI4L, Fig. 4 and ECTV-DEVM002, Fig. 6). We have also shown the usefulness of fluorescent sorting strategies to rapidly enrich for the recombinant virus population by subjecting the initial transfection/infection reaction to FACS analysis (Fig. 2A). Alternatively, a more traditional approach of using fluorescent microscopy to identify and pick individual plaques expressing YFP can be used. The flexibility of the SEM system in the purification of recombinant poxviruses is particularly important when working with highly attenuated strains. Importantly, we show here that even the double deleted DI4L/DF4L VV strains, which have severe replication defects and exhibit a small plaque phenotype [23], can be produced and purified using the SEM system. We had hypothesized that expression of cytoplasmic Cre- recombinase would maximize recombination efficiency since poxviruses replicate in the cytoplasm. Indeed a single passage of the VV-DTk-yfp-gpt virus on U2OS cells expressing cytoplasmic Cre is sufficient to eliminate nearly all of the yfp-gpt cassette from the viral genome (Fig. 2B and 2C). However, infection of U2OS cells stably expressing nuclear Cre still led to nearly 100% recombination efficiency (Fig. 2C). Not unlike other proteins, the Cre enzyme is initially located in the cytoplasm prior to transport to the nucleus. We speculate that in cells selected for high levels of Cre expression, the amount of enzyme is not limiting for the excision reaction and thus even small amounts, or temporary cytoplasmic expression of Cre from nuclear-cre expressing cells may be sufficient to facilitate recombination. Importantly, despite lower Cre-expression levels achieved from transient transfection of U2OS cells with pMC-Cre (Fig. 1D), the recombination frequency was still .40% (Fig. 2C). These data illustrate how the SEM system can be used with any transfectable cell line while maintaining the efficiency of the Cre/loxP reaction. To confirm that the residual DNA signature was consistent for all recombinant viruses post Cre recombination, DNA sequencing was performed at the insertion site (Figs. 3C, 4D, 6D). In all cases, one 34 base pair loxP site remains in the viral genome, with some remnants of the respective SEM cloning vectors. We have observed no obvious impact of retention of this 34 base pair sequence on virus replication and indeed this has been studied PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24643