Poxvirus Vector Development Figure 2. FACS purification of recombinant VV-DTk infected cells and Cre-recombinase mediated removal of the selectable marker. (A) Dot plot of YFP fluorescence versus side scatter from Fluorescence Activated Cell Sorting (FACS) analysis of U2OS cells mock infected, or infected with a mixture of parental VV and recombinant VV virus expressing YFP. (B) Purified recombinant VV-DTk-yfp-gpt-1loxP (control virus) or VV-DTk-yfp- gpt virus were used to infect stable cytoplasmic Cre-expressing cells (U2OS-Cre) or parental U2OS cells in 6-well plates, and were monitored for foreign gene expression (firefly luciferase) and marker gene expression (YFP-GPT fusion protein). (C) Percent YFP-negative VV-DTk’ plaques on U2OS cells after passage of virus on Cre cells (nuclear or cytoplasmic stable cell lines or transiently transfected U2OS cells). (D) Map of the pSEM-1 plasmid indicating the primer pairs used in the PCR reactions to characterize the genome of recombinant VV-DTk viruses shown in panel E. (E) PCR analysis of DNA extracted from VV-DTk-yfp-gpt-1loxP (control virus), VV-DTk-yfp-gpt and VV-DTk’ viruses. The PCR products span: i. across the left Tk flanking region, ii. across the luciferase gene, iii. across the yfp-gpt selectable marker, iv. across the right the Tk flanking region. n.s = not-significant, * p = 0.001. doi:10.1371/journal.pone.0024643.g002 revealed a consensus sequence with no conflicts (Fig. 3C). As predicted, the residual DNA signature contained 1 loxP site and remnants from the pSEM-1 vector. Construction of the second generation SEM cloning vector and the VV-DI4L viruses The second generation SEM plasmid, termed pDGloxPKO was designed with multiple cloning sites flanking the yfp-gpt cassette. This permits insertion of homologous targeting sequences flanking the selectable marker and/or therapeutic transgene. This vector was also designed such that the yfp-gpt cassette and its early/late viral promoter are flanked by loxP sites. To test the importance of loxP site orientation during Cre-recombination, two pDGloxPKO vectors were created with the loxP sites in either the same orientation (pDGloxPKODEL) (Figure S2A), or oriented towards each other (pDGloxPKOINV) (Figure S2B). Inserting homolo- gous sequences of DNA from the VV genome flanking the I4L gene (I3L and I5L homology) into both pDGloxPKODEL and pDGloxPKOINV vectors generated pDGloxPKODEL-DI4L and pDGloxPKOINV-DI4L, respectively (Fig. 4A and 4B). These vectors were used to create two strains of recombinant VV in which the I4L locus is disrupted by vector-derived yfp-gpt cassette sequences (see schematic in Fig. 4C). Using vector pDGloxPKODEL- DI4L, which contains identically oriented loxP sites flanking the PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24643