encoding sequences to the left of the EVM002 gene. Similarly the PCR and primers EVM002-39 (left) plus EVM002-39 (right) (Table S1) were used to produce a 150 bp product encoding sequences to the right of the EVM002 gene in the ectromelia virus (ECTV) genome. These two 150 bp PCR fragments were cloned into pGEM-T vectors (Promega). The fragment encoding sequences located on the left side of the EVM002 gene was subsequently cloned into the pDGloxPKODEL vector using the XhoI and HindIII restriction sites to create pDGloxPKO-EVM002- 59. Finally, the EVM002-39 150 bp was cloned into pDGloxPKO- EVM002-59 using the BamHI and NotI restriction sites to create pDGloxPKODEL-DEVM002. Cloning – Cre plasmids The gene encoding a form of Cre protein lacking a nuclear localization sequence (NLS) was PCR amplified from pMC-Cre using Taq polymerase and primers NLS-free Cre (left) and NLS- free Cre (right) (Table S1). The PCR product was digested with XhoI and NotI and ligated into pLPCX cut with the same enzymes. This produced plasmid pLPCX-Cyto_Cre. A Cre plasmid was also created with the NLS intact by digesting Cre from pMC-Cre, and inserting the gene into the XhoI site of the pLXSN vector to form pLXSN-Nuc_Cre. U2OS-Cre cells Retroviral transduction methods were used to produce cells expressing nuclear- or cytoplasmic-localized Cre protein. A stock of transducing particles was first produced by using calcium phosphate to co-transfect 293T cells with 7 mg of plasmid DNA (either pLPCX-Cyto_Cre or pLSXN-Nuc_Cre) along with the retroviral helper plasmids pHIT60 and pHIT456 [45]. Virus- containing supernatants were harvested two days later and the debris removed with a 0.45-mm filter. To perform the transduction experiment, 2.56105 U2OS cells were infected with 2 mL of the virus for 2 hr in the presence of 8 mg/mL of polybrene. Two days later, G418 (800 mg/mL) or puromycin (1 mg/mL) were added and drug-resistant recombinants were harvested a week later. Purified recombinant poxviruses were passaged 1-to-3 times in these cells to remove loxP-flanked genes. Recombinant viruses Viruses constructed from the pSEM-1 vector were produced from the VV strain Wyeth [7]. U2OS cells were infected with VV at a multiplicity of infection (MOI) of 0.01 and then transfected with plasmid pSEM-1 or pSEM-1ctrl vectors using Lipofectamine 2000 (Invitrogen). The cells were incubated at 37uC for 4 hr, the medium was replaced, and then the cells cultured for two more days. The dishes were harvested, YFP-positive cells sorted using flow cytometry, and the YFP-positive population purified further by selecting YFP-positive plaques in the presence of MPA for 2 rounds of purification. This produced VV strain VV-DTk- yfp-gpt with both loxP sites in the same orientation and VV strain VV- DTk-yfp-gpt-1loxP bearing a single loxP site. Viruses bearing deletions of the I4L gene were produced using pDGloxPKOINV-DI4L and pDGloxPKODEL-DI4L vectors in VV strain Western Reserve (WR), and in DF4L VV strains [23]. BSC- 40 cells were infected for 1 hr (MOI = 2), transfected with plasmid DNA using Lipofectamine 2000, and virus harvested two days later. Recombinant virus were isolated using three rounds of plaque purification on BSC-40 cells in MPA selection media (DMEM supplemented with 10% serum, 50 U/mL penicillin, 50 mg/mL of streptomycin, 200 mM glutamine, 250 mg/mL xanthine, 15 mg/mL hypoxanthine, and 25 mg/mL MPA). Poxvirus Vector Development A recombinant ECTV was constructed by infecting BGMK cells with ECTV (strain Moscow) at an MOI = 0.01, followed by transfection with XhoI and NotI linearized pDGloxPKO- DEVM002 DNA. The virus was harvested two days later and foci were purified twice in the presence of MPA and twice in the absence of MPA selection. Cell sorting and flow cytometry U2OS cells were infected with virus at MOI ,0.1 for 6 hr, and sorted for YFP fluorescence on a MoFlo cytometer (DakoCytoma- tion). The top 5% of YFP positive cells (approximately 3,000 cells) were collected and mixed with ,300,000 uninfected U2OS cells and cultured for two days in a 10 cm dish. For flow cytometry, ,106 HeLa cells were infected with virus at a MOI = 3 for 4 hr. The cells were suspended in 0.5 mL of PBS containing 1% FBS, and YFP fluorescence determined using a FACScan flow cytometer (Becton-Dickinson) and CellQuest software (Version 3.1). Luciferase and fluorescence detection using IVIS Imager The fluorescence and bioluminescence were detected using a 200 Series IVIS Imager (Xenogen) and Living ImageH v2.5 software. Cell lysates from Cre cell lines infected with VV-DTk- yfp-gpt were used to infect U2OS cells which were first imaged for YFP fluorescence prior to incubation with 20 mg of D-luciferin (Molecular Imaging Products Company) added directly to the cultured cells. D-luciferin was incubated with the cells for 20 min at 37uC. Identical program settings (exposure, aperture and pixel binning) were used for all experiments. Quantification of Cre recombination efficiency U2OS cells stably expressing nuclear or cytoplasmic versions of U2OS, or U2OS cells transiently transfected with Cre recombi- nase were infected at an MOI of 0.05 for 2 days. Virus progeny was then analyzed by U2OS plaque assay for YFP expression of individual virus plaques. Fluorescence microscopy To detect YFP, 26105 BGMK cells were seeded on coverslips and infected (or mock infected) with virus at a MOI = 0.01 for two days. The cells were fixed with 2% paraformaldehyde, mounted in 50% glycerol containing 4 mg/mL N-propyl-gallate (Sigma- Aldrich), and 250 mg/mL 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen), and visualized using a Zeiss 710 confocal microscope equipped with Zeiss Zen software 2009, light edition. For immunofluorescence microscopy, 56104 Cre-expressing U2OS cells were plated per 1.8 cm2 well in Nunc Chamber SlidesH. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were treated for 30 min with blocking buffer (0.2% Triton X-100, 5% normal goat serum in PBS) and then incubated overnight at 4uC with a 1:100 diluted anti-Cre antibody in blocking buffer. The cells were washed with PBS, incubated for 30 min with Cy3– conjugated goat anti-rabbit antibody (Jackson), washed three times, and then mounted on a slide. DNA was stained with 1.5 mg/mL 4,6-diamidino-2-phenylindole (DAPI). Cell images were captured using a Zeiss Axioplan 2 microscope equipped with Axioview 3.1 software. Virus DNA extraction DNA was extracted from sucrose cushion purified virus stocks according to methods adapted from Meyer et al. [46]. Briefly, 200 mL of purified virus stock was treated with lysis buffer (1.4 mL PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24643