recombination of the homologous sequences between the vector and the viral genome [21]. Using traditional approaches, the frequency of recombination is typically less than 0.1% [22], and the isolation of purified recombinant virus is tedious and time- consuming. Recombinant poxviruses are often attenuated, and have reduced growth kinetics and plaque size compared to their wild type counterparts [23]. Historically, the target site of choice has been VV thymidine kinase (Tk), but any non-essential locus can be modified or disrupted in this manner. Recombinants are then isolated and plaque purified. A number of selection methods have been described including selection for Tk-positive or negative phenotypes [21], and resistance to neomycin [24] or mycophenolic acid (MPA) [25]. One can also use plaque assays to identify viruses encoding b-galactosidase [26], b-glucuronidase [27], or fluores- cent reporter constructs [28]. Although these methods work well and greatly facilitate the recovery of recombinant viruses, the use of selectable markers inevitably results in the creation of a product that contains genetic information with no therapeutic value. Recombinant poxvirus therapeutics would be considered safer vectors (most notably in the view of regulatory agencies), if the selectable markers were removed from the poxvirus genome [29]. Furthermore, the expression of marker genes from recombinant poxviruses may affect the overall fitness of the virus. Demmin et al. have shown that the expression levels of neighboring genes can be affected by the highly active transcription of marker genes incorporated into other large DNA viruses [30]. To facilitate the removal of selectable markers, Falkner and colleagues [31,32] developed transient selection methods wherein a selectable marker is flanked by tandem DNA repeats. The virus is stable while under selection, however the marker is lost through VV dependent homologous recombination once the selection pressure is removed. A similar system was employed by Alejo et al., to create ECTV recombinants [33]. Although these authors describe efficiencies in excess of 90% for removal of the selectable marker, the recombination reaction is a random and lengthy process that relies on poxvirus machinery, and involves more than six rounds of purification. Typically, the efficiency of poxvirus recombination is quite low [22], recombinant viruses are often attenuated and hard to propagate in the presence of wild-type virus [34], and many time-consuming rounds of plaque purifica- tion are needed to isolate the desired final viral product. An improved technology is needed that allows for the specific, controlled and efficient removal of selectable markers from recombinant poxvirus genomes. Here we define a new methodological tool for rapidly producing marker-free recombinant poxviruses. This improved vector development system, which we have termed Selectable and Excisable Marker (SEM), takes advantage of the well-character- ized Cre/loxP site-specific recombination system to efficiently excise the reporter gene [35,36]. This strategy avoids the many rounds of passage that are required when using virus recombina- tion systems to excise genes flanked by tandemly duplicated elements [31,32,33]. The SEM system offers the convenience of positive selection of recombinants using both fluorescent and/or drug-based strategies. Since recombinant poxvirus therapeutics are often created with multiple gene knockouts (or knock-ins), the SEM system was designed to be re-usable, therefore eliminating the use of additional reporter genes that would otherwise complicate and lengthen the overall cloning and selection processes. To demonstrate the efficiency and utility of the method, we have applied the SEM strategy to generate viruses with targeted disruptions of different genes using two different poxvirus species. Our results suggest that the SEM vector development Poxvirus Vector Development system will not only be useful for the creation of novel poxvirus therapeutics, but also for basic virological studies. Results Characterization of the components of the SEM system The Selectable and Excisable Marker system is summarized in Fig. 1A. The first generation transfer vector pSEM-1 encoded a foreign gene (firefly luciferase), a selectable marker as a fusion between yellow fluorescent protein (yfp) and guanine phosphoribosyltransfer- ase (gpt) genes, and loxP sites in the same orientation flanking the selectable marker, with the target insertion site as the VV Tk locus (Fig. 1B). To confirm that the expression of YFP was not disrupted from the creation of the YFP-GPT fusion protein, YFP expression was analyzed by western blot from U2OS cells either mock-transfected (lane 1), or transiently transfected with either pEYFP-C1 as a positive control (lane 2) or plasmids containing the yfp-gpt fused gene (pEYFP-gpt or pEYFP-gpt-1loxP) in lanes 3 and 4, respectively (Figure S1A). The YFP and YFP-GPT fusion proteins have predicted molecular weights of 26 and 45.5 kDa respectively. The cellular distribution of Cre from Cre recombinase- expressing cell lines was analyzed by immunofluorescence from parental U2OS cells (control), nuclear Cre cells (Nuc-Cre), and cytoplasmic Cre (Cyto-Cre) cells illustrating that the absence of the nuclear localization sequence in the Nuc-Cre cells leads to accumulation of the enzyme in the cytoplasm (Fig. 1C). Cre expression levels were compared among mock-transfected U2OS, U2OS cells transiently transfected with pMC-Cre, and both stable cell lines (Fig. 1D). In cells that were transiently or stably expressing Cre-recombinase, anti-Cre western blot analysis identified a band at 35 kDa, corresponding to the Cre enzyme. The expression of Cre in the stable cell lines was 2-fold greater when compared to the transiently transfected cells. Isolation of a marker-free Tk-deleted, luciferase- expressing VV using the SEM system (YFP)-based fluorescence activated cell sorting (FACS) was performed on mock infected U2OS cells as a control, and U2OS cells that had been infected with a mixture of parental VV (Wyeth strain), and recombinant VV generated from pSEM-1 expressing the YFP-GPT fusion protein (VV-DTk-yfp-gpt) (Fig. 2A). To ensure the highest possible FACS stringency, a cut-off of 0.5% background fluorescence was maintained by comparing the mock- infected U2OS cells to the recombinant YFP-expressing VV infected U2OS cells (235 positive background cells from 47,421 U2OS cells counted, compared to 3004 positive recombinant VV infected cells, from 49,498 U2OS counted). These sorted cells were then mixed with uninfected U2OS cells, plated into multi- well dishes, and subjected to two more rounds of YFP+ plaque purification. To promote fast and simple removal of the yfp-gpt cassette from recombinant viruses generated using the SEM system, viruses were passaged on a U2OS cell line expressing a cytoplasmic form of Cre recombinase. VV-DTk-yfp-gpt-1loxP (control virus) and VV-DTk- yfp-gpt recombinant VV were passaged on either parental U2OS cells, or U2OS cells stably expressing cytoplasmic Cre recombi- nase (U2OS-Cre) (Fig. 2B). The U2OS cells were monitored for both YFP fluorescence and for luciferase-mediated biolumines- cence using the IVIS Imager (Xenogen) and Living ImageH v2.5 software. As shown in the top half of Fig. 2B, both VV-DTk-yfp- gpt-1loxP control virus, and VV-DTk-yfp-gpt viruses express luciferase and YFP when used to infect parental U2OS cells. Infection of U2OS-Cre cells by the VV-DTk-yfp-gpt-1loxP and PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24643