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Construction of Human Cytomegalovirus Mutants with Markerless BAC Mutagenesis

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Human Cytomegaloviruses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2244))

Abstract

To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they are studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of these loss-of-function viral mutants to the wild-type virus allow for the identification of the relevance and role for a particular gene in the viral replication cycle. Targeted CMV mutagenesis has made huge advances over the past 20 years. The cloning of CMV genomes into Escherichia coli as bacterial artificial chromosomes (BAC) allows for not only quick and efficient deletion of viral genomic regions, individual genes, or single-nucleotide exchanges in the viral genome but also the insertion of heterologous genetic sequences for gain-of-function approaches. The conceptual advantage of this strategy is that it overcomes the restrictions of recombinant technologies in cell culture systems. Namely, recombination in infected cells occurs only in a few clones, and their selection is not possible if the targeted genes are relevant for virus replication and are not able to compete for growth against the unrecombined parental viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, providing selective growth advantage to the recombined genomes and thus clonal selection of viruses with even extremely poor fitness. Here we describe the methods used for the generation of a CMV BAC, targeted mutagenesis of BAC clones, and transfection of human cells with CMV BAC DNA in order to reconstitute the viral infection process.

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Authors and Affiliations

  1. Department of Vaccinology, Helmholtz Centre for Infection Research, Braunschweig, Germany

    M. Zeeshan Chaudhry & Luka Čičin-Šain

  2. Institute of Virology, Hannover Medical School, Hannover, Germany

    Martin Messerle

  3. Center for Individualized Infection Medicine (CIIM), A Joint Venture of HZI and MHH, Braunschweig, Germany

    Luka Čičin-Šain

Authors
  1. M. Zeeshan Chaudhry
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  2. Martin Messerle
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  3. Luka Čičin-Šain
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Corresponding author

Correspondence to Luka Čičin-Šain .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Immunology, Louisiana State University Health Sciences Center—Shreveport, Shreveport, LA, USA

    Andrew D. Yurochko

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Chaudhry, M.Z., Messerle, M., Čičin-Šain, L. (2021). Construction of Human Cytomegalovirus Mutants with Markerless BAC Mutagenesis. In: Yurochko, A.D. (eds) Human Cytomegaloviruses. Methods in Molecular Biology, vol 2244. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1111-1_8

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  • DOI: https://doi.org/10.1007/978-1-0716-1111-1_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1110-4

  • Online ISBN: 978-1-0716-1111-1

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