A Selectable and Excisable Marker System for the Rapid
`Creation of Recombinant Poxviruses
`Julia L. Rintoul1,2.
`, Jiahu Wang2.
`, Don B. Gammon3, Nicholas J. van Buuren3, Kenneth Garson2, Karen
`Jardine2, Michele Barry3, David H. Evans3, John C. Bell1,2*
`
`1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada, 2 Centre for Cancer Therapeutics, Ottawa
`Hospital Research Institute, Ottawa, Canada, 3 Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, Li Ka Shing Institute of Virology,
`University of Alberta, Edmonton, Canada
`
`Abstract
`
`Background:Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a
`number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses
`often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes
`however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.
`
`Methodology/Principal Findings: Here we describe an improved strategy that allows for the creation of marker-free
`recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion
`marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent
`removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into
`vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation
`during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into
`the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here
`with the creation of an ectromelia virus recombinant lacking the EVM002 gene.
`
`Conclusion/Significance: The system described here thus provides a faster, simpler and more efficient means to create
`clinic-ready recombinant poxviruses for therapeutic gene therapy applications.
`
`Citation: Rintoul JL, Wang J, Gammon DB, van Buuren NJ, Garson K, et al. (2011) A Selectable and Excisable Marker System for the Rapid Creation of Recombinant
`Poxviruses. PLoS ONE 6(9): e24643. doi:10.1371/journal.pone.0024643
`
`Editor: William P. Halford, Southern Illinois University School of Medicine, United States of America
`
`Received December 23, 2010; Accepted August 16, 2011; Published September 8, 2011
`Copyright: ß 2011 Rintoul et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
`unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
`
`Funding: This work was supported by grants from the National Cancer Institute of Canada, Terry Fox Foundation, Natural Sciences Engineering Research Council,
`and the Canadian Institutes of Health Research to JB, DE and MB. JLR was supported by a CIHR Doctoral award: Frederick Banting and Charles Best Canada
`Graduate Scholarship. JW was supported by a Lymphoma Research Foundation of Canada Research Fellowship. DBG was supported by a Ralph Steinhauer Award
`of Distinction from the Province of Alberta. NJVB was supported by a Doctoral NSERC Canada Graduate Scholarship and an Alberta Heritage Foundation for
`Medical Research Incentive Award. MB holds a Tier I Canada Research Chair, a Howard Hughes International Scholar Award and an Alberta Heritage for Medical
`Research Senior Scholar award. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
`
`Competing Interests: The authors have declared that no competing interests exist.
`
`* E-mail: jbell@ohri.ca
`. These authors contributed equally to this work.
`
`Introduction
`
`Poxviruses comprise a large family of double-stranded DNA
`viruses that infect a wide range of hosts. Vaccinia virus (VV) is the
`prototypic member of the Orthopoxvirus genus and the best-studied
`virus in the poxvirus family. Since the eradication of smallpox [1],
`VV and other poxvirus species have continued to be used for the
`treatment of human disease [2,3] in part because a greater
`understanding of poxvirus biology has led to safer and more
`efficacious poxvirus-based therapeutics. The poxvirus genome is
`easily genetically modified and can accommodate inserts exceed-
`ing 25 kb [4] using strategies that are dependent upon virus-
`encoded homologous recombination [5,6]. Using these approach-
`es, recombinant VV has since proven to be valuable as a vector for
`gene
`therapy
`in a number
`of
`therapeutic
`applications
`[4,7,8,9,10,11,12,13,14,15]. Similarly, other members of
`the
`poxvirus family have also been explored for their potential as
`viral vectors for therapeutic purposes [9,10,16,17]. Genetically
`
`from other
`immunogens
`that express
`engineered poxviruses
`infectious agents have shown some promise as novel vaccines
`against diseases like acquired immunodeficiency syndrome [11],
`malaria [12],
`tuberculosis [18], and cancer [7,8,10,13]. As a
`cancer vaccine, poxviruses have the potential to generate a strong
`anti-tumoural
`immune response, especially when genetically
`modified to express cytokines like IL-2 [14] or cell surface
`receptors like CD70 that are indicative of oncogenic transforma-
`tion [15]. Lastly, poxviruses have been successfully engineered as
`oncolytic agents, offering the advantage of a strong anti-tumoural
`immune response combined with cancer cell-specific replication
`[7,16,17,19,20]. A number of these poxvirus candidates have
`advanced to human clinical trials [10,11,12,13,19], highlighting
`the therapeutic potential of poxvirus recombinants.
`Poxvirus recombinants are typically produced by constructing a
`plasmid containing the gene(s) of
`interest
`flanked by DNA
`sequences homologous to the desired target locus, followed by
`transfection of the plasmid into VV infected cells to allow for
`
`PLoS ONE | www.plosone.org
`
`1
`
`September 2011 | Volume 6 |
`
`Issue 9 | e24643
`
`Exhibit #
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`Bell 1103
`
`9/23/22
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`exhibitsticker.com
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`Page 1 of 13
`
`

`

`A Selectable and Excisable Marker System for the Rapid
`Creation of Recombinant Poxviruses
`Julia L. Rintoul1,2.
`, Jiahu Wang2.
`, Don B. Gammon3, Nicholas J. van Buuren3, Kenneth Garson2, Karen
`Jardine2, Michele Barry3, David H. Evans3, John C. Bell1,2*
`
`1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada, 2 Centre for Cancer Therapeutics, Ottawa
`Hospital Research Institute, Ottawa, Canada, 3 Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, Li Ka Shing Institute of Virology,
`University of Alberta, Edmonton, Canada
`
`Abstract
`
`Background:Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a
`number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses
`often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes
`however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.
`
`Methodology/Principal Findings: Here we describe an improved strategy that allows for the creation of marker-free
`recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion
`marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent
`removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into
`vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation
`during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into
`the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here
`with the creation of an ectromelia virus recombinant lacking the EVM002 gene.
`
`Conclusion/Significance: The system described here thus provides a faster, simpler and more efficient means to create
`clinic-ready recombinant poxviruses for therapeutic gene therapy applications.
`
`Citation: Rintoul JL, Wang J, Gammon DB, van Buuren NJ, Garson K, et al. (2011) A Selectable and Excisable Marker System for the Rapid Creation of Recombinant
`Poxviruses. PLoS ONE 6(9): e24643. doi:10.1371/journal.pone.0024643
`
`Editor: William P. Halford, Southern Illinois University School of Medicine, United States of America
`
`Received December 23, 2010; Accepted August 16, 2011; Published September 8, 2011
`Copyright: ß 2011 Rintoul et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
`unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
`
`Funding: This work was supported by grants from the National Cancer Institute of Canada, Terry Fox Foundation, Natural Sciences Engineering Research Council,
`and the Canadian Institutes of Health Research to JB, DE and MB. JLR was supported by a CIHR Doctoral award: Frederick Banting and Charles Best Canada
`Graduate Scholarship. JW was supported by a Lymphoma Research Foundation of Canada Research Fellowship. DBG was supported by a Ralph Steinhauer Award
`of Distinction from the Province of Alberta. NJVB was supported by a Doctoral NSERC Canada Graduate Scholarship and an Alberta Heritage Foundation for
`Medical Research Incentive Award. MB holds a Tier I Canada Research Chair, a Howard Hughes International Scholar Award and an Alberta Heritage for Medical
`Research Senior Scholar award. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
`
`Competing Interests: The authors have declared that no competing interests exist.
`
`* E-mail: jbell@ohri.ca
`. These authors contributed equally to this work.
`
`Introduction
`
`Poxviruses comprise a large family of double-stranded DNA
`viruses that infect a wide range of hosts. Vaccinia virus (VV) is the
`prototypic member of the Orthopoxvirus genus and the best-studied
`virus in the poxvirus family. Since the eradication of smallpox [1],
`VV and other poxvirus species have continued to be used for the
`treatment of human disease [2,3] in part because a greater
`understanding of poxvirus biology has led to safer and more
`efficacious poxvirus-based therapeutics. The poxvirus genome is
`easily genetically modified and can accommodate inserts exceed-
`ing 25 kb [4] using strategies that are dependent upon virus-
`encoded homologous recombination [5,6]. Using these approach-
`es, recombinant VV has since proven to be valuable as a vector for
`gene
`therapy
`in a number
`of
`therapeutic
`applications
`[4,7,8,9,10,11,12,13,14,15]. Similarly, other members of
`the
`poxvirus family have also been explored for their potential as
`viral vectors for therapeutic purposes [9,10,16,17]. Genetically
`
`from other
`immunogens
`that express
`engineered poxviruses
`infectious agents have shown some promise as novel vaccines
`against diseases like acquired immunodeficiency syndrome [11],
`malaria [12],
`tuberculosis [18], and cancer [7,8,10,13]. As a
`cancer vaccine, poxviruses have the potential to generate a strong
`anti-tumoural
`immune response, especially when genetically
`modified to express cytokines like IL-2 [14] or cell surface
`receptors like CD70 that are indicative of oncogenic transforma-
`tion [15]. Lastly, poxviruses have been successfully engineered as
`oncolytic agents, offering the advantage of a strong anti-tumoural
`immune response combined with cancer cell-specific replication
`[7,16,17,19,20]. A number of these poxvirus candidates have
`advanced to human clinical trials [10,11,12,13,19], highlighting
`the therapeutic potential of poxvirus recombinants.
`Poxvirus recombinants are typically produced by constructing a
`plasmid containing the gene(s) of
`interest
`flanked by DNA
`sequences homologous to the desired target locus, followed by
`transfection of the plasmid into VV infected cells to allow for
`
`PLoS ONE | www.plosone.org
`
`1
`
`September 2011 | Volume 6 |
`
`Issue 9 | e24643
`
`Exhibit #
`
`Bell 1103
`
`9/23/22
`
`exhibitsticker.com
`
`Page 2 of 13
`
`

`

`recombination of the homologous sequences between the vector
`and the viral genome [21]. Using traditional approaches, the
`frequency of recombination is typically less than 0.1% [22], and
`the isolation of purified recombinant virus is tedious and time-
`consuming. Recombinant poxviruses are often attenuated, and
`have reduced growth kinetics and plaque size compared to their
`wild type counterparts [23]. Historically, the target site of choice
`has been VV thymidine kinase (Tk), but any non-essential locus
`can be modified or disrupted in this manner. Recombinants are
`then isolated and plaque purified. A number of selection methods
`have been described including selection for Tk-positive or negative
`phenotypes [21], and resistance to neomycin [24] or mycophenolic
`acid (MPA) [25]. One can also use plaque assays to identify viruses
`encoding b-galactosidase [26], b-glucuronidase [27], or fluores-
`cent reporter constructs [28].
`Although these methods work well and greatly facilitate the
`recovery of recombinant viruses, the use of selectable markers
`inevitably results in the creation of a product that contains genetic
`information with no therapeutic value. Recombinant poxvirus
`therapeutics would be considered safer vectors (most notably in the
`view of regulatory agencies),
`if
`the selectable markers were
`removed from the poxvirus genome [29]. Furthermore,
`the
`expression of marker genes from recombinant poxviruses may
`affect the overall fitness of the virus. Demmin et al. have shown that
`the expression levels of neighboring genes can be affected by the
`highly active transcription of marker genes incorporated into other
`large DNA viruses [30].
`To facilitate the removal of selectable markers, Falkner and
`colleagues [31,32] developed transient selection methods wherein
`a selectable marker is flanked by tandem DNA repeats. The virus
`is stable while under selection, however the marker is lost through
`VV dependent homologous recombination once the selection
`pressure is removed. A similar system was employed by Alejo et al.,
`to create ECTV recombinants [33]. Although these authors
`describe efficiencies in excess of 90% for removal of the selectable
`marker, the recombination reaction is a random and lengthy
`process that relies on poxvirus machinery, and involves more than
`six rounds of purification. Typically, the efficiency of poxvirus
`recombination is quite low [22], recombinant viruses are often
`attenuated and hard to propagate in the presence of wild-type
`virus [34], and many time-consuming rounds of plaque purifica-
`tion are needed to isolate the desired final viral product. An
`improved technology is needed that allows
`for the specific,
`controlled and efficient removal of
`selectable markers
`from
`recombinant poxvirus genomes.
`Here we define a new methodological tool for rapidly producing
`marker-free recombinant poxviruses. This
`improved vector
`development
`system, which we have termed Selectable and
`Excisable Marker (SEM), takes advantage of the well-character-
`ized Cre/loxP site-specific recombination system to efficiently
`excise the reporter gene [35,36]. This strategy avoids the many
`rounds of passage that are required when using virus recombina-
`tion systems to excise genes flanked by tandemly duplicated
`elements [31,32,33]. The SEM system offers the convenience of
`positive selection of recombinants using both fluorescent and/or
`drug-based strategies. Since recombinant poxvirus therapeutics
`are often created with multiple gene knockouts (or knock-ins), the
`SEM system was designed to be re-usable, therefore eliminating
`the use of additional
`reporter genes
`that would otherwise
`complicate and lengthen the overall cloning and selection
`processes. To demonstrate the efficiency and utility of the method,
`we have applied the SEM strategy to generate viruses with
`targeted disruptions of different genes using two different poxvirus
`species. Our results suggest that the SEM vector development
`
`Poxvirus Vector Development
`
`system will not only be useful for the creation of novel poxvirus
`therapeutics, but also for basic virological studies.
`
`Results
`
`Characterization of the components of the SEM system
`The Selectable and Excisable Marker system is summarized in
`Fig. 1A. The first generation transfer vector pSEM-1 encoded a
`foreign gene (firefly luciferase), a selectable marker as a fusion
`between yellow fluorescent protein (yfp) and guanine phosphoribosyltransfer-
`ase (gpt) genes, and loxP sites in the same orientation flanking the
`selectable marker, with the target insertion site as the VV Tk locus
`(Fig. 1B). To confirm that
`the expression of YFP was not
`disrupted from the creation of the YFP-GPT fusion protein, YFP
`expression was analyzed by western blot from U2OS cells either
`mock-transfected (lane 1), or transiently transfected with either
`pEYFP-C1 as a positive control (lane 2) or plasmids containing the
`yfp-gpt fused gene (pEYFP-gpt or pEYFP-gpt-1loxP) in lanes 3 and
`4, respectively (Figure S1A). The YFP and YFP-GPT fusion
`proteins have predicted molecular weights of 26 and 45.5 kDa
`respectively.
`The cellular distribution of Cre from Cre recombinase-
`expressing cell lines was analyzed by immunofluorescence from
`parental U2OS cells (control), nuclear Cre cells (Nuc-Cre), and
`cytoplasmic Cre (Cyto-Cre) cells illustrating that the absence of the
`nuclear localization sequence in the Nuc-Cre cells
`leads
`to
`accumulation of the enzyme in the cytoplasm (Fig. 1C). Cre
`expression levels were compared among mock-transfected U2OS,
`U2OS cells transiently transfected with pMC-Cre, and both stable
`lines (Fig. 1D). In cells that were transiently or stably
`cell
`expressing Cre-recombinase, anti-Cre western blot analysis
`identified a band at 35 kDa, corresponding to the Cre enzyme.
`The expression of Cre in the stable cell lines was 2-fold greater
`when compared to the transiently transfected cells.
`
`Isolation of a marker-free Tk-deleted, luciferase-
`expressing VV using the SEM system
`(YFP)-based fluorescence activated cell sorting (FACS) was
`performed on mock infected U2OS cells as a control, and U2OS
`cells that had been infected with a mixture of parental VV (Wyeth
`strain), and recombinant VV generated from pSEM-1 expressing
`the YFP-GPT fusion protein (VV-DTk-yfp-gpt)
`(Fig. 2A). To
`ensure the highest possible FACS stringency, a cut-off of 0.5%
`background fluorescence was maintained by comparing the mock-
`infected U2OS cells to the recombinant YFP-expressing VV
`infected U2OS cells (235 positive background cells from 47,421
`U2OS cells counted, compared to 3004 positive recombinant VV
`infected cells, from 49,498 U2OS counted). These sorted cells
`were then mixed with uninfected U2OS cells, plated into multi-
`+
`plaque
`well dishes, and subjected to two more rounds of YFP
`purification.
`To promote fast and simple removal of the yfp-gpt cassette from
`recombinant viruses generated using the SEM system, viruses were
`passaged on a U2OS cell line expressing a cytoplasmic form of Cre
`recombinase. VV-DTk-yfp-gpt-1loxP (control virus) and VV-DTk-
`yfp-gpt recombinant VV were passaged on either parental U2OS
`cells, or U2OS cells stably expressing cytoplasmic Cre recombi-
`nase (U2OS-Cre) (Fig. 2B). The U2OS cells were monitored for
`both YFP fluorescence and for luciferase-mediated biolumines-
`cence using the IVIS Imager (Xenogen) and Living ImageH v2.5
`software. As shown in the top half of Fig. 2B, both VV-DTk-yfp-
`gpt-1loxP control virus, and VV-DTk-yfp-gpt viruses express
`luciferase and YFP when used to infect parental U2OS cells.
`Infection of U2OS-Cre cells by the VV-DTk-yfp-gpt-1loxP and
`
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`Poxvirus Vector Development
`
`Figure 1. Overview of the poxvirus Selectable and Excisable Marker cloning system. (A) Schematic illustrating the Selectable and
`Excisable Marker poxvirus cloning system.
`(B) First generation poxvirus cloning vector pSEM-1 with labeled open reading frames.
`(C)
`Immunofluorescence detection of Cre recombinase from U2OS cell lines expressing nuclear or cytoplasmic Cre, or control U2OS cells. (D) Western
`blot analysis of Cre recombinase from U2OS cells mock transfected (U2OS), transiently transfected (pMC-Cre) or stably expressing Cre recombinase
`targeted to either the nucleus or the cytoplasm (Nuc-Cre and Cyto-Cre respectively).
`doi:10.1371/journal.pone.0024643.g001
`
`VV-DTk-yfp-gpt viruses led to strong luciferase transgene expres-
`sion, however expression of YFP is only detectable from cells
`infected with the VV-DTk-yfp-gpt-1loxP virus. This illustrates the
`qualitative efficiency by which Cre-expressing U2OS cells excise
`loxP-flanked markers, as well as the stability of the transgene
`expression during the recombination reaction. To quantitatively
`determine the efficiency of the Cre recombination reaction, U2OS
`cells expressing Cre recombinase were infected with the VV-DTk-
`yfp-gpt virus. Virus progeny was then analyzed for the percent YFP
`positive versus negative virus plaques by U2OS plaque assay. As
`seen in Figure 2C, both cytoplasmic and nuclear Cre expression
`from stable cell lines resulted in nearly 100% efficiency for marker
`gene excision, whereas transient transfection of Cre recombinase
`was only 44% efficient.
`The genomic composition of the VV recombinant viruses was
`analyzed by PCR of viral DNA before and after removal of the yfp-
`gpt cassette. A schematic of the virus genome at the Tk insertion
`site is shown (Fig. 2D) to illustrate the primer pairs used in the
`analysis. Primers were designed to amplify regions of DNA at the
`insertion site (i and iv), surrounding the luciferase transgene (ii),
`and across the yfp-gpt selectable marker (iii). For three of the PCR
`reactions (primer pairs i, ii, and iv), the amplicons for each of the
`viruses tested were the same and show that the Cre recombination
`reaction does not affect the genome structure outside of the 2loxP
`region (ii), and that the Tk locus was the site of homologous
`recombination (i, iv) (Fig. 2E). Prior to passage on Cre-expressing
`cells, PCR using primer pair (iii) of DNA from both VV-DTk-yfp-
`gpt-1loxP and VV-DTk-yfp-gpt viruses produced bands at 1900 and
`2000 bp, respectively. The 100 bp difference can be attributed to
`
`the lack of one loxP site in the VV-DTk-yfp-gpt-1loxP virus. PCR of
`DNA from the marker-free recombinant VV-DTk’ virus using
`primer pair (iii), produced a much smaller band due to Cre-
`mediated deletion of the yfp-gpt gene.
`
`Genomic analysis and confirmation of identity of 3
`independent clones of the VV-DTk-yfp-gpt and VV-DTk’
`viruses
`The genomic composition of the VV-DTk-yfp-gpt and VV-DTk’
`viruses was analyzed by restriction enzyme digestion, southern blot
`hybridization and sequencing analysis at the Cre-recombination
`site (Fig. 3). Three independent clones of the VV-DTk-yfp-gpt virus
`were compared to the parental VV Wyeth strain, before and after
`passage on Cre-expressing cells. The DNA fragment containing
`the Tk insertion site is highlighted (arrow) in the digest of the
`the yfp-gpt cassette
`parental VV Wyeth virus.
`Interestingly,
`included a HindIII restriction site. This led to a unique DNA
`for the VV-DTk-yfp-gpt clones
`(Fig. 3A). The DNA
`digest
`fragment containing the Tk insertion site is disrupted in digests
`of the VV-DTk-yfp-gpt viruses, and is represented by unique bands
`at ,6000 and ,2800 bp (see arrows, Fig. 3A). Digests of the VV-
`DTk’ clones resemble the parental VV digest, since the yfp-gpt
`cassette was removed by Cre-recombination. Importantly, the
`southern hybridization for yfp demonstrates that there was only 1
`insertion site of yfp-gpt during poxvirus homologous recombination
`(Fig. 3B). DNA sequence analysis of the 3 VV-DTk’ clones was
`performed to illustrate the consistency of
`the residual DNA
`signature following Cre-recombination. A DNAStar sequence
`the 3 VV-DTk’ clones
`alignment of
`the DNA from each of
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`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
`into both pDGloxPKODEL and
`gene (I3L and I5L homology)
`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
`
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`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
`
`Page 6 of 13
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`

`

`Poxvirus Vector Development
`
`Figure 4. Creation of the VV-DI4Lmutants 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
`loxP flanked inserts (Fig. 5B, right
`different arrangements of
`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