Poxvirus Vector Development Figure 3. Confirmation of genomic composition of 3 independent recombinant VV-DTk viruses. (A) An ethidium bromide stained DNA gel of genomic HindIII restriction digests of viral DNA isolated from parental VV (Wyeth Strain), 3 clones of VV-DTk-yfp-gpt and 3 clones of VV-DTk’. Arrows indicate the Tk insertion site (VV-Wyeth), and the unique bands that result from insertion of the yfp-gpt cassette (VV-DTk-yfp-gpt). (B) Southern hybridization of the DNA gel in A identifying the yfp insert present in the genome of the VV-DTk-yfp-gpt clones, but not in parental VV-Wyeth or the VV-DTk’ clones. (C) DNAStar sequence alignment at the yfp-gpt insertion site of DNA isolated from the 3 VV-DTk’ clones post Cre passage. doi:10.1371/journal.pone.0024643.g003 yfp-gpt cassette led to the generation of strain VV-DI4LDEL, whereas using vector pDGloxPKOINV-DI4L in which the inserted yfp-gpt cassette is flanked by loxP sites oriented towards each other generated virus VV-DI4LINV. The genomic composition of the VV-DI4LDEL virus post Cre passage was analyzed by sequencing at the excision site of yfp-gpt and revealed the Cre/loxP signature remaining in the viral genome post Cre-recombination (Fig. 4D). As expected, the virus contains 1 loxP site, and remnants from the pDGloxPKO vector. To exclude the possibility that there were multiple yfp-gpt insertion sites, the entire genome of VV- DI4LDEL virus post Cre passage was sequenced. The sequence of the VV-DI4LDEL recombinant was compared to the sequence of the parental VV (Western Reserve) using a dotplot analysis (Figure S3A). These data illustrate that the VV-DI4LDEL virus is disrupted only at the I4L locus, thereby confirming 1 insertion site at the desired locus. Cre-mediated recombination of viral DNA is dependent upon loxP site orientation Both the pDGloxPKODEL and pDGloxPKOINV plasmids were used to explore the dependence of loxP orientation on Cre-mediated recombination of poxvirus genomes. Previous work has shown that Cre-mediated recombination between identically-oriented loxP sites generates deletions while oppositely oriented loxP sites lead to inversion of DNA sequences [35,36], but it remained to be formally proven that this would also hold-true in virus-infected cells. Recombinant DI4L viruses were analyzed by PCR for the presence of the I4L gene before (BSC-40 passage) and after (U2OS-Cre passage) Cre recombination (Fig. 5A). As an additional test of the SEM approach, we have included F4L-inactivated recombinants in these experiments, as DF4L virus backgrounds have been reported to have severe growth kinetics compared to their wild-type counterpart [23]. Referring to the expected amplicon sizes and primer pairs illustrated in Fig. 4C, the PCR products generated using primers flanking the yfp-gpt cassette (A+D) exhibited sizes indicative of replacement of the I4L gene with the yfp-gpt cassette in viruses cultured in BSC-40 cells (Fig. 5A, left panel). Upon passage in Cre- expressing cells, only those viruses produced using pDGloxPKODEL vectors (DI4LDEL, and DI4L/DF4LDEL) exhibited the deletion of the yfp-gpt cassette (Fig. 5A, right panel). This shows that Cre-mediated deletions only occur when the DNA is flanked by identically oriented loxP sites. The orientation of the yfp-gpt cassette in recombinant viruses produced using the pDGloxPKOINV-DI4L vector were also analyzed by PCR before and after passage through Cre-expressing cells. We used primer pairs designed to amplify the yfp-gpt cassette PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24643