Generation of Modified Pestiviruses by Targeted Recombination

Thomas Bruun Rasmussen, Martin Barfred Friis, Peter Christian Risager, Ilona Reimann, Martin Beer

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    Abstract

    Infectious cDNA clones are a prerequisite for directed genetic manipulation of pestivirus RNA genomes. We have developed a novel strategy to facilitate manipulation and rescue of modified pestiviruses from infectious cDNA clones based on bacterial artificial chromosomes (BACs). The strategy involves targeted modification of viral cDNA genomes, cloned within BACs, by Red/ET recombination-mediated mutagenesis in E.coli DH10B cells. Using recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused in principal on any sequence within the viral genome and hence is not limited to the use of internal restriction sites. Rescue of modified pestiviruses can be obtained by electroporation of cell cultures with full-length RNA transcripts in vitro transcribed from the recombined BAC clones. We have used this approach to generate a series of new pestivirus BACs modified within different genomic regions and infectious pestiviruses have been rescued from several of these new constructs, demonstrating that recombination-mediated mutagenesis of pestivirus BACs provides a useful tool for expediting the construction of recombinant pestiviruses.
    Original languageEnglish
    Publication date2011
    Publication statusPublished - 2011
    Event8th ESVV Pestivirus Symposium - Hannover, Germany
    Duration: 25 Sep 201128 Sep 2011
    Conference number: 8
    http://www.pestivirus2011.de/

    Conference

    Conference8th ESVV Pestivirus Symposium
    Number8
    CountryGermany
    CityHannover
    Period25/09/201128/09/2011
    Internet address

    Cite this

    Rasmussen, T. B., Friis, M. B., Risager, P. C., Reimann, I., & Beer, M. (2011). Generation of Modified Pestiviruses by Targeted Recombination. Abstract from 8th ESVV Pestivirus Symposium, Hannover, Germany. http://www.pestivirus2011.de/