Abstract
Poxvirus expression vectors have gained widespread use for expression of foreign proteins and as delivery vehicles for vaccine antigens. We have developed a novel method using the poxvirus as a library vector for functional selection of specific cDNA. Poxviruses have several unique and useful properties as a library vector. Most importantly, because poxviruses are packaged into fully infectious particles in the cell cytoplasm, specific recombinants can be readily recovered even from a very small number of selected cells. Moreover, in contrast to libraries constructed in retrovirus or plasmid-based vectors, recombinant vaccinia virus can be efficiently recovered even from cells that have been induced to undergo apoptosis or cessation of cell growth. In the past, the major obstacle in this application to poxviruses has been the low frequency with which recombinants can be generated. The most commonly used method to construct recombinant poxvirus is homologous recombination. The frequency of recombinants derived in this manner is of the order of 0.1%, sufficient to recover a recombinant of a purified DNA clone in a transfer plasmid, but far too low to permit construction of a representative cDNA library. We have developed a method that generates nearly 100% recombinant vaccinia viruses at good titer. We have termed this method trimolecular recombination. cDNA libraries of as many as 107 or more independent viral recombinants can be constructed by trimolecular recombination. For the first time, large, diverse, and representative cDNA libraries can be screened in a vaccinia virus-based expression vector.
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References
Moss, B. (1991) Vaccinia virus: A tool for research and vaccine development. Science 252, 1662–1667.
Merchlinsky, M., Eckert, D., Smith, E., and Zauderer, M. (1997) Construction and characterization of vaccinia direct ligation vectors. Virology 238, 444–451.
Somogyi, P., Frazier, J., and Skinner, M. (1993) Fowlpox virus host range restriction: Gene expression, DNA replication, and morphogenesis in nonpermissive mammalian cells. Virology 197, 439–444
Scheiflingler, F., Dorner, F., and Falkner, F. (1992) Construction of chimeric vaccinia virus by molecular cloning and packaging. Proc. Natl. Acad. Sci. USA 89, 9977–9981.
Smith, E. S., Mandokhot, A., Evans, E. E., et al. (2001) Lethality-based selection of recombinant genes in mammalian cells: Application to identifying tumor antigens. Nature Med. 7, 967–972.
Earl, P. L. and Moss, B. (1991) Generation of recombinant vaccinia viruses, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds.), Greene/Whiley-Interscience, New York, pp. 16.17.1–16.17.16.
In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells. US Patent application no. 20020123057, September 5, 2002 (international patent application no. WO 02102855, December 27, 2002).
Method of producing or identifying intrabodies in eukaryotic cells. US patent application no. 10/052,942, January 23, 2002 (international patent application WO 02086096, October 31, 2002).
T cells specific for target antigens and vaccines based thereon. US patent application no.08/935,377, September 22, 1997 (international patent application no. WO 0028016, May 18, 2000).
Methods of producing a library and methods of selecting polynucleotides of interest. US patent application no. 20030022157, January 30, 2003 (international patent application no. WO 0172995, October 4, 2001).
Methods of identifying regulator molecules. US patent application no. 20020192675, December 19, 2002 (international patent application no. WO 02062822, August 15, 2002).
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© 2004 Humana Press Inc., Totowa, NJ
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Smith, E.S., Shi, S., Zauderer, M. (2004). Construction of cDNA Libraries in Vaccinia Virus. In: Isaacs, S.N. (eds) Vaccinia Virus and Poxvirology. Methods in Molecular Biology, vol 269. Humana Press. https://doi.org/10.1385/1-59259-789-0:065
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DOI: https://doi.org/10.1385/1-59259-789-0:065
Publisher Name: Humana Press
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