Poxvirus Vector Development Figure 1. Overview of the poxvirus Selectable and Excisable Marker cloning system. (A) Schematic illustrating the Selectable and Excisable Marker poxvirus cloning system. (B) First generation poxvirus cloning vector pSEM-1 with labeled open reading frames. (C) Immunofluorescence detection of Cre recombinase from U2OS cell lines expressing nuclear or cytoplasmic Cre, or control U2OS cells. (D) Western blot analysis of Cre recombinase from U2OS cells mock transfected (U2OS), transiently transfected (pMC-Cre) or stably expressing Cre recombinase targeted to either the nucleus or the cytoplasm (Nuc-Cre and Cyto-Cre respectively). doi:10.1371/journal.pone.0024643.g001 VV-DTk-yfp-gpt viruses led to strong luciferase transgene expres- sion, however expression of YFP is only detectable from cells infected with the VV-DTk-yfp-gpt-1loxP virus. This illustrates the qualitative efficiency by which Cre-expressing U2OS cells excise loxP-flanked markers, as well as the stability of the transgene expression during the recombination reaction. To quantitatively determine the efficiency of the Cre recombination reaction, U2OS cells expressing Cre recombinase were infected with the VV-DTk- yfp-gpt virus. Virus progeny was then analyzed for the percent YFP positive versus negative virus plaques by U2OS plaque assay. As seen in Figure 2C, both cytoplasmic and nuclear Cre expression from stable cell lines resulted in nearly 100% efficiency for marker gene excision, whereas transient transfection of Cre recombinase was only 44% efficient. The genomic composition of the VV recombinant viruses was analyzed by PCR of viral DNA before and after removal of the yfp- gpt cassette. A schematic of the virus genome at the Tk insertion site is shown (Fig. 2D) to illustrate the primer pairs used in the analysis. Primers were designed to amplify regions of DNA at the insertion site (i and iv), surrounding the luciferase transgene (ii), and across the yfp-gpt selectable marker (iii). For three of the PCR reactions (primer pairs i, ii, and iv), the amplicons for each of the viruses tested were the same and show that the Cre recombination reaction does not affect the genome structure outside of the 2loxP region (ii), and that the Tk locus was the site of homologous recombination (i, iv) (Fig. 2E). Prior to passage on Cre-expressing cells, PCR using primer pair (iii) of DNA from both VV-DTk-yfp- gpt-1loxP and VV-DTk-yfp-gpt viruses produced bands at 1900 and 2000 bp, respectively. The 100 bp difference can be attributed to the lack of one loxP site in the VV-DTk-yfp-gpt-1loxP virus. PCR of DNA from the marker-free recombinant VV-DTk’ virus using primer pair (iii), produced a much smaller band due to Cre- mediated deletion of the yfp-gpt gene. Genomic analysis and confirmation of identity of 3 independent clones of the VV-DTk-yfp-gpt and VV-DTk’ viruses The genomic composition of the VV-DTk-yfp-gpt and VV-DTk’ viruses was analyzed by restriction enzyme digestion, southern blot hybridization and sequencing analysis at the Cre-recombination site (Fig. 3). Three independent clones of the VV-DTk-yfp-gpt virus were compared to the parental VV Wyeth strain, before and after passage on Cre-expressing cells. The DNA fragment containing the Tk insertion site is highlighted (arrow) in the digest of the parental VV Wyeth virus. Interestingly, the yfp-gpt cassette included a HindIII restriction site. This led to a unique DNA digest for the VV-DTk-yfp-gpt clones (Fig. 3A). The DNA fragment containing the Tk insertion site is disrupted in digests of the VV-DTk-yfp-gpt viruses, and is represented by unique bands at ,6000 and ,2800 bp (see arrows, Fig. 3A). Digests of the VV- DTk’ clones resemble the parental VV digest, since the yfp-gpt cassette was removed by Cre-recombination. Importantly, the southern hybridization for yfp demonstrates that there was only 1 insertion site of yfp-gpt during poxvirus homologous recombination (Fig. 3B). DNA sequence analysis of the 3 VV-DTk’ clones was performed to illustrate the consistency of the residual DNA signature following Cre-recombination. A DNAStar sequence alignment of the DNA from each of the 3 VV-DTk’ clones PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24643