extensively in mammalian systems where the retention of loxP sequences has no impact on mRNA stability, promoter activity or genome integrity [37,38]. To further validate the consistency of the recombination events of the SEM system, 3 clones of the VV- DTk virus pre (VV-DTk-yfp-gpt) and post (VV-DTk’) Cre passage were analyzed by HindIII DNA restriction digest and southern hybridization (Fig. 3). As predicted, the DNA signatures are consistent, and the Tk locus is the only site of poxvirus recombination. Since the VV-DI4L and ECTV-DEVM002’ recombinant viruses were purified as heterogeneous populations (MPA drug selection), the sequence analysis is representative of all recombination events. To confirm that there was only 1 site of yfp- gpt integration in the VV-DI4LDEL virus, the entire genome of the virus was sequenced. A dotplot comparison of recombinant VV- DI4LDEL to parental VV illustrates that the recombination event was targeted to the I4L locus (Figure S3). Using the second generation SEM cloning vectors (pDGloxP- KODEL and pDGloxPKOINV), we have shown for the first time that similar to recombination of cellular DNA [35,36], Cre- mediated recombination between identically-oriented loxP sites generates deletions, while oppositely oriented loxP sites leads to inversions of viral DNA sequences (Fig. 5). Interestingly, targeted inversions of the yfp-gpt cassette when generating viruses using the pDGloxPKOINV-DI4L targeting vector identified an additional application for the SEM system. Site-specific inversions may be useful for transcriptional interference studies [39] in which a poxvirus promoter could drive the same mRNA from opposite strands. This approach would allow one to assess the potential strand-specific influence of neighbouring transcriptional processes and read-through transcripts on the efficiency of transcription of the reporter mRNA. In summary, the SEM strategy provides an efficient means to ‘‘surgically’’ manipulate viral genomes by adding or deleting genes without leaving unwanted marker genes behind. We have demonstrated the applicability of the SEM system to create recombinant viruses in two different species, targeting three different poxvirus loci by deletions and/or insertions, including strains with severe replication defects. By inclusion of fluorescence and drug selection strategies, the SEM system offers both flexibility and simplicity for the purification of recombinant viruses. The yfp- gpt cassette was also shown to be useful during Cre-recombination to monitor efficiency, which could be enhanced even further by making use of a reverse gpt selection strategy [40]. This system will be especially useful when multiple deletions or transgenes are to be added sequentially to a single vector. Finally, the tools developed in the SEM system may also contribute to basic virology research, like those that involve transcriptional interference studies. Materials and Methods Antibodies and reagents The following primary antibodies were used: rabbit anti-GFP/ YFP (Abcam), Cre (Novagen), VV I4 (a gift from Dr. C. Mathews, Oregon State University), mouse anti-b-actin (Sigma-Aldrich) and VV I3 [41]. Secondary antibodies conjugated to horseradish peroxidase (BioRad) or infrared dyes (Li-Cor) were used to detect primary antibodies in immunoblotting. OptiMEM (Invitrogen) was used for transfection experiments. Cells The following cells were purchased from American Type Tissue Collection: human osteosarcoma (U2OS), human embryonic kidney, large T antigen transformed (293T), and green monkey kidney (BSC-40) cells. Buffalo green monkey kidney (BGMK) cells Poxvirus Vector Development were purchased from Diagnostic Hybrid. Cell lines were maintained in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% fetal calf serum (Cansera), penicillin (100 U/ml), and streptomycin sulfate (100 mg/ml) at 37uC in 5% CO2. All cell lines were tested and found clear of mycoplasma contamination. Plasmids Plasmids pEYFP-C1, pLPCX, and pLXSN were purchased from Clontech and pGEM-T was purchased from Promega. Plasmid pMC-Cre [42] was a gift from Dr. Klaus Rajewsky (Harvard Medical School), pSEL-eGFP [20] and pSC65-luc [43] were gifts from Dr. David Bartlett (University of Pittsburgh) and pHIT60 and pHIT456 were gifts from Dr. Ian Lorimer (University of Ottawa). Cloning – pSEM-1 vector The gpt gene was first excised from pSEL-eGFP with BseY1, repaired, and ligated into a pT7blue-3 cloning vector (Novagen). The gpt gene was then excised with SalI and BamHI and ligated, in frame, into XhoI/BamHI cut pEYFP-C1. This created pEYFP-gpt bearing a fusion of the two polypeptides. The first loxP sequence was constructed by annealing together two oligonucleotides with the same sequence (loxP, Table S1), using the In-Fusion-2 method (Clontech) [44]. The pEYFP-gpt plasmid was then cut with NheI and recombined with the double-stranded DNA loxP site to obtain ploxP-EYFP-gpt. The second loxP sequence was inserted in a similar way by cutting ploxP-EYFP-gpt at the BamHI site, and inserting the double-stranded oligonucleotide loxP (Table S1). The resulting plasmid, p2loxP-EYFP-gpt, contained two loxP sites with the same orientation flanking the gene encoding the YFP-GPT fusion protein. The pSEM-1 plasmid was assembled by inserting the loxP-yfp-gpt- loxP cassette into pSC65-luc. This was accomplished by cutting p2loxP-EYFP-gpt and pSC65-luc plasmids with XhoI and BamHI restriction enzymes, and ligating the loxP-yfp-gpt-loxP insert into the VV Tk gene in pSC65-luc. Two vectors were created: a negative control vector containing only the first loxP site (pSEM-1ctrl), and a vector containing two loxP sites inserted in the same orientation and flanking the reporter construct (pSEM-1) (Fig. 1B). Cloning – pDGloxPKO vectors A second series of vectors were assembled using gene synthesis (GeneArt) and contained two multiple cloning sites flanking the loxP sites. To test the importance of loxP orientation, two versions of these plasmids were created with the loxP sites either in the same (pDGloxPKODEL) or in the opposite orientation (pDGloxP- KOINV) with respect to one another (Figure S2). Cloning – pDGloxPKO-DI4L vectors The PCR and primers I5L (left) plus I5L (right) (Table S1) were used to prepare a ,430 bp PCR product encoding sequences flanking the ‘‘I5L’’ side of the VV I4L locus. Similarly the PCR and primers I3L (left) plus I3L (right) (Table S1) were used to produce a ,340 bp PCR product containing sequences flanking the ‘‘I3L’’ side of the I4L locus. These DNAs were then cloned into the SpeI/SalI (I5L homology) and SacII/BglII (I3L homology) restriction sites of pDGloxPKOINV and pDGloxPKO- DEL. Adding these sequences created vectors pDGloxPKOINV- DI4L and pDGloxPKODEL-DI4L (Fig. 4). Cloning – pDGloxPKODEL-DEVM002 vector The PCR and primers EVM002-59 (left) plus EVM002-59 (right) (Table S1) were used to prepare a 150 bp product PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24643