Abstract
Traditional methods for genetic manipulation of poxviruses rely on low-frequency natural recombination in virus-infected cells. Although these powerful systems represent the technical foundation of current knowledge and applications of poxviruses, they require long (≥500 bp) flanking sequences for homologous recombination, an efficient viral selection method, and burdensome, time-consuming plaque purification. The beginning of the twenty-first century has seen the application of bacterial artificial chromosome (BAC) technology to poxviruses as an alternative method for their genetic manipulation, following the invention of a long-sought-after method for deriving a BAC clone of vaccinia virus (VAC-BAC) by Arban Domi and Bernard Moss. The key advantages of the BAC system are the ease and versatility of performing genetic manipulation using bacteriophage λ Red recombination (recombineering), which requires only ∼50 bp homology arms that can be easily created by PCR, and which allows seamless mutations lacking any marker gene without having to perform transient-dominant selection. On the other hand, there are disadvantages, including the significant setup time, the risk of contamination of the cloned genome with bacterial insertion sequences, and the nontrivial issue of removal of the BAC cassette from derived viruses. These must be carefully weighed to decide whether the use of BACs will be advantageous for a particular application, making pox-BAC systems likely to complement, rather than supplant, traditional methods in most laboratories.
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References
Messerle M, Crnkovic I, Hammerschmidt W, Ziegler H, Koszinowski UH (1997) Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci USA 94:14759–14763
Domi A, Moss B (2002) Cloning the vaccinia virus genome as a bacterial artificial chromosome in Escherichia coli and recovery of infectious virus in mammalian cells. Proc Natl Acad Sci USA 99:12415–12420
Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361–388
Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4:206–223
Domi A, Moss B (2005) Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage lambda-based recombination. Nat Methods 2:95–97
Cottingham MG, Andersen RF, Spencer AJ, Saurya S, Furze J, Hill AV, Gilbert SC (2008) Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA). PLoS One 3:e1638
Meisinger-Henschel C, Spath M, Lukassen S, Wolferstatter M, Kachelriess H, Baur K, Dirmeier U, Wagner M, Chaplin P, Suter M, Hausmann J (2010) Introduction of the six major genomic deletions of Modified Vaccinia Virus Ankara (MVA) into the parental vaccinia virus is not sufficient to reproduce an MVA-like phenotype in cell culture and in mice. J Virol 84:9907–9919
Roth SJ, Hoper D, Beer M, Feineis S, Tischer BK, Osterrieder N (2011) Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC). Vet Res 42:3
Domi A, Weisberg AS, Moss B (2008) Vaccinia virus E2L null mutants exhibit a major reduction in extracellular virion formation and virus spread. J Virol 82:4215–4226
Osborne JD, Da Silva M, Frace AM, Sammons SA, Olsen-Rasmussen M, Upton C, Buller RM, Chen N, Feng Z, Roper RL, Liu J, Pougatcheva S, Chen W, Wohlhueter RM, Esposito JJ (2007) Genomic differences of vaccinia virus clones from Dryvax smallpox vaccine: the Dryvax-like ACAM2000 and the mouse neurovirulent Clone-3. Vaccine 25:8807–8832
Scheiflinger F, Dorner F, Falkner FG (1992) Construction of chimeric vaccinia viruses by molecular cloning and packaging. Proc Natl Acad Sci USA 89:9977–9981
Cottingham MG, Gilbert SC (2010) Rapid generation of markerless recombinant MVA vaccines by en passant recombineering of a self-excising bacterial artificial chromosome. J Virol Methods 168:233–236
Tischer BK, Kaufer BB, Sommer M, Wussow F, Arvin AM, Osterrieder N (2007) A self-excisable infectious bacterial artificial chromosome clone of varicella-zoster virus allows analysis of the essential tegument protein encoded by ORF9. J Virol 81:13200–13208
Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33:e36
Yao XD, Evans DH (2004) Construction of recombinant vaccinia viruses using leporipoxvirus-catalyzed recombination and reactivation of orthopoxvirus DNA. Methods Mol Biol 269:51–64
Merchlinsky M, Moss B (1992) Introduction of foreign DNA into the vaccinia virus genome by in vitro ligation: recombination-independent selectable cloning vectors. Virology 190:522–526
Tsung K, Yim JH, Marti W, Buller RM, Norton JA (1996) Gene expression and cytopathic effect of vaccinia virus inactivated by psoralen and long-wave UV light. J Virol 70:165–171
Wong QN, Ng VC, Lin MC, Kung HF, Chan D, Huang JD (2005) Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli. Nucleic Acids Res 33:e59
DeVito JA (2008) Recombineering with tolC as a selectable/counter-selectable marker: remodeling the rRNA operons of Escherichia coli. Nucleic Acids Res 36:e4
Tischer BK, Smith GA, Osterrieder N (2011) En passant mutagenesis: a two step markerless red recombination system. Methods Mol Biol 634:421–430
Bal TR, Anand B, Yogeeswari P, Sriram D (2005) Synthesis and evaluation of anti-HIV activity of isatin beta-thiosemicarbazone derivatives. Bioorg Med Chem Lett 15:4451–4455
Mayr A, Malicki K (1966) Attenuation of virulent fowl pox virus in tissue culture and characteristics of the attenuated virus. Zentralbl Veterinarmed B 13:1–13
Cresawn SG, Prins C, Latner DR, Condit RC (2007) Mapping and phenotypic analysis of spontaneous isatin-beta-thiosemicarbazone resistant mutants of vaccinia virus. Virology 363:319–332
Posfai G, Plunkett G 3rd, Feher T, Frisch D, Keil GM, Umenhoffer K, Kolisnychenko V, Stahl B, Sharma SS, de Arruda M, Burland V, Harcum SW, Blattner FR (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044–1046
Acknowledgments
The author would like to thank Dr Richard Wade-Martins, Department of Anatomy and Human Genetics, University of Oxford, UK for supplying the protocol upon which that in Subheading 3.2 is based; Michaela Späth, Kay Brinkmann, and Jürgen Hausman from Bavarian-Nordic GmbH, Martinsreid, Germany for the protocol in Subheading 3.4; Dr Michael Skinner, Imperial College London, UK for agreeing to supply FP9; and principal investigators Prof. Adrian V. S. Hill and Dr Sarah C. Gilbert, Jenner Institute, University of Oxford, UK.
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Cottingham, M.G. (2012). Genetic Manipulation of Poxviruses Using Bacterial Artificial Chromosome Recombineering. In: Isaacs, S. (eds) Vaccinia Virus and Poxvirology. Methods in Molecular Biology, vol 890. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-876-4_3
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DOI: https://doi.org/10.1007/978-1-61779-876-4_3
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