54% sucrose, 15 mL 2-mercaptoethanol, 50 mL if 20 mg/mL, proteinase-K and 250 mL 10% SDS) for 4 hours at 55uC. The samples were then purified by phenol extraction. Sequencing of viral DNA Viral DNA from Cre-passaged viruses was subjected to PCR amplification at the site of Cre recombination. Sequencing was performed using the Applied Biosystem DNA analyzers. The entire genome of both parental VV Western Reserve and recombinant VV-DI4LDEL virus post Cre passage were sequenced from 5 mg of extracted viral DNA at the Genome Que´bec Innovation Centre (Montre´al, Que´bec) using a high throughput pyrosequencing approach on a Roche 454 GS FLX Titanium sequencer platform. CLC Genomics Workbench 4.6 software was used to assemble the genome sequence. The viral genomes were analyzed and compared using dotplots prepared using the program Gepard [47] with default settings. DNA restriction digest and southern hybridization Viral DNA was analyzed using Southern blot. Briefly, 7 mg of DNA was digested overnight with HindIII restriction enzyme (Invitrogen) at 37uC. DNA digests were electrophoresed on a 0.8% agarose gel in 16 TAE buffer at 46 V for 6 hours, then stained with ethidium bromide and photographed. Gels were first denatured for 45 mins, then neutralized for 45 mins prior to transfer over night onto Hybond-N membrane (Amersham), and finally UV cross-linked. A 931 bp yfp-gpt probe was labeled with P32 a-dCTP (PerkinElmer) using the Multiprime DNA Labeling kit (Amersham). The blot was blocked for 2 hours at 42uC in a formamide pre-hybridization buffer followed by over night hybridization with 5 mCurries of denatured probe. Membranes were washed and exposed to a phospho screen for 18 hours and imaged on a Storm imaging system (Storm 860; Molecular Dynamics) using ImageQuant software, version 5.2. Western blotting For immunoblotting, cells were lysed in RIPA buffer with protease inhibitors (CompleteH, Roche) for 30 min on ice. The cell lysates were sonicated for 30 seconds, centrifuged at 10,0006g and the cell pellets discarded, fractionated by electrophoresis on a Nupage 4–12% Bis-Tris gel (Invitrogen), transferred to Hybond-C extra nitrocellulose membranes (Amersham), and probed with the indicated primary antibodies. Secondary antibodies conjugated to horseradish peroxidase or Li-Cor infrared dyes were used to detect bound antigens. References 1. Fenner F, Henderson D. A, Arita L, Jezek Z, Ladnyi ID, eds. Smallpox and Its Eradication. Geneva: W.H.O. 2. Gilbert PA, McFadden G (2006) Poxvirus cancer therapy. Recent Pat Antiinfect Drug Discov 1: 309–321. 3. Moroziewicz D, Kaufman HL (2005) Gene therapy with poxvirus vectors. Curr Opin Mol Ther 7: 317–325. 4. Smith GL, Moss B (1983) Infectious poxvirus vectors have capacity for at least 25 000 base pairs of foreign DNA. Gene 25: 21–28. 5. Fenner F, Comben BM (1958) [Genetic studies with mammalian poxviruses. I. Demonstration of recombination between two strains of vaccina virus.]. Virology 5: 530–548. 6. Nakano E, Panicali D, Paoletti E (1982) Molecular genetics of vaccinia virus: demonstration of marker rescue. Proc Natl Acad Sci U S A 79: 1593– 1596. 7. Mastrangelo MJ, Maguire HC, Jr., Eisenlohr LC, Laughlin CE, Monken CE, et al. (1999) Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma. Cancer Gene Ther 6: 409–422. 8. Erbs P, Findeli A, Kintz J, Cordier P, Hoffmann C, et al. (2008) Modified vaccinia virus Ankara as a vector for suicide gene therapy. Cancer Gene Ther 15: 18–28. Poxvirus Vector Development Statistical Analysis Data were analyzed by a one-way Anova, followed by a Turkey post-hoc for multiple group comparison at a 99% confidence interval. Supporting Information Figure S1 Detection of YFP protein expression by western blot analysis. (A) Western blotting of transfected cells. Anti-YFP western blot of U2OS cells mock transfected (1), or transfected with plasmid DNA pEYFP-c1 (2), pEYFP-gpt, (3), or pEYFP-gpt-1loxP (4). (TIFF) Figure S2 Plasmid maps of pDGloxPKODEL and pDGloxP- KOINV. (A) Map of cloning vector pDGloxPKODEL and (B) pDGloxPKOINV with labeled open reading frames. (TIFF) Figure S3 Sequencing analysis of genomic viral DNA from VV-DI4LDEL. (A) Dotplot comparison of sequenced viral genomes of wild type Western Reserve vaccinia virus and recombinant VV-DI4LDEL virus post Cre passage using default settings available in Gepard [47]. (TIFF) Primers used in the study. Table S1 (DOC) Acknowledgments Thank you to Mr. Paul Oleynik from the StemCore laboratories for assistance in generating the FACS data. Thank you to Ms. Megan Desaulniers for preparing Western Reserve vaccinia virus, and Ms. Li Qin for preparing genomic viral DNA for sequencing analysis. Thank you to Dr. Christopher Mathews for the anti-VV I4 antibodies. Thank you to Dr. Ian Lorimer for the pHIT60 and pHIT456 plasmids. Thank you to Dr. Klaus Rajewsky for sharing the pMC-Cre plasmid, and Dr. David Bartlett for sharing the pSC65-luc plasmid. Thank you to Dr. John Barrett for his technical expertise. Thank you to Catherine Mathis (Transgene, France) for her expertise and communications regarding Regulatory Guidelines for poxvirus recombinants. Author Contributions Conceived and designed the experiments: JLR JW DBG NJVB KG KJ MB DHE JCB. Performed the experiments: JLR JW DBG NJVB KG. Analyzed the data: JLR JW DBG NJVB KG KJ MB DHE JCB. Contributed reagents/materials/analysis tools: JLR JW DBG NJVB KG KJ MB DHE JCB. Wrote the paper: JLR JW JCB. 9. Cadoz M, Strady A, Meignier B, Taylor J, Tartaglia J, et al. (1992) Immunisation with canarypox virus expressing rabies glycoprotein. Lancet 339: 1429–1432. 10. Kantoff PW, Schuetz TJ, Blumenstein BA, Glode LM, Bilhartz DL, et al. (2010) Overall survival analysis of a phase II randomized controlled trial of a Poxviral- based PSA-targeted immunotherapy in metastatic castration-resistant prostate cancer. J Clin Oncol 28: 1099–1105. 11. Kanesa-thasan N, Smucny JJ, Hoke CH, Marks DH, Konishi E, et al. (2000) Safety and immunogenicity of NYVAC-JEV and ALVAC-JEV attenuated recombinant Japanese encephalitis virus–poxvirus vaccines in vaccinia-nonim- mune and vaccinia-immune humans. Vaccine 19: 483–491. 12. Bejon P, Ogada E, Mwangi T, Milligan P, Lang T, et al. (2007) Extended follow- up following a phase 2b randomized trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya. PLoS One 2: e707. 13. Harrop R, Drury N, Shingler W, Chikoti P, Redchenko I, et al. (2007) Vaccination of colorectal cancer patients with modified vaccinia ankara encoding the tumor antigen 5T4 (TroVax) given alongside chemotherapy induces potent immune responses. Clin Cancer Res 13: 4487–4494. 14. Dasgupta S, Bhattacharya-Chatterjee M, O’Malley BW, Jr., Chatterjee SK (2006) Recombinant vaccinia virus expressing interleukin-2 invokes anti-tumor PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24643