Poxvirus Vector Development Figure 4. Creation of the VV-DI4L mutants from the second generation SEM cloning vectors. (A) Map of cloning vector pDGloxPKODEL- DI4L and (B) pDGloxPKOINV-DI4L with labeled open reading frames. (C) Schematic displaying the strategy for knock-out of the I4L open reading frame from VV strain WR, and possible outcomes of Cre-recombination of recombinant VV-DI4L virus generated from either the pDGloxPKODEL-DI4L or pDGloxPKOINV-DI4L vectors. (D) DNA sequence analysis of the VV-DI4LDEL virus post Cre passage. doi:10.1371/journal.pone.0024643.g004 in the forward orientation (pairs A+B, C+D), and also in the case of yfp-gpt inversions (pairs A+C, B+D). The viruses isolated from BSC-40 cells produced PCR products consistent with a single orientation identical to that seen in original plasmid (Fig. 5B, left panel). However, upon passage through Cre-expressing cells, the PCR products displayed a pattern characteristic of a mix of two different arrangements of loxP flanked inserts (Fig. 5B, right panel). Western blot analysis was used to confirm deletion of the I4L locus of all DI4L strains. Recombinant DI4L viruses produced with either pDGloxPKOINV-DI4L or pDGloxPKODEL-DI4L vectors led to inactivation of I4 protein expression and this inactivation was specific since I3, (expressed from the neighbouring gene, I3L) levels remained unchanged (Fig. 5C). The YFP-GPT protein was only deleted from strains that had been generated with the pDGloxPKODEL-DI4L targeting vector and passaged in Cre- expressing cells (Fig. 5C, right panel), further confirming that deletion events need both identically-oriented loxP sites and exposure to Cre activity. Collectively, these results demonstrate that the SEM vector system can be used to either delete or invert sequences within the viral genome, upon passage of the selected strains in Cre-expressing cells. The SEM system can be used with other members of the Poxvirus family The SEM system was also used to create a recombinant ECTV (strain Moscow) lacking the EVM002 gene using the transfer plasmid pDGloxPKODEL-DEVM002 (Fig. 6A). Referring to the schematic outlined in Fig. 6B, ECTV viral recombination with pDGloxPKODEL-DEVM002 with subsequent MPA drug selection created the primary recombinant ECTV-DEVM002-yfp-gpt. The virus was passaged through U2OS-Cre cells to obtain the marker- free recombinant virus ECTV-DEVM002’. The genomic compo- sition of wild type ECTV (ECTV-wt), ECTV-DEVM002-yfp-gpt, and ECTV-DEVM002’ viruses was confirmed by PCR analysis (Fig. 6C). These PCR analyses demonstrate at the genomic level the deletion of the EVM002 gene, the insertion of the yfp-gpt cassette, and its subsequent removal following Cre-mediated recombination. The genomic composition of the ECTV- DEVM002’ was analyzed by sequencing at the excision site of yfp-gpt to illustrate the Cre/loxP signature remaining in the viral genome post recombination (Fig. 6D). As expected, the virus contains 1 loxP site, and remnants from the pDGloxPKO vector. YFP protein expression from the ECTV viruses was also analyzed by confocal microscopy (Fig. 6E). Mock-infected BGMK cells PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24643