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Use of Recombinant Approaches to Construct Human Cytomegalovirus Mutants

  • Iryna Dekhtiarenko
  • Luka Čičin-ŠainEmail author
  • Martin Messerle
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1119)

Abstract

To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they be studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of loss of function viral mutants to the wild-type virus allow 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 15 years. The cloning of CMV genomes into (E. coli) as bacterial artificial chromosomes (BAC) allows 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 viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, allowing a selective growth advantage to the recombined genomes. 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.

Key words

Bacterial artificial chromosome (BAC) Targeted mutagenesis Homologous recombination Antibiotic selection 

Notes

Acknowledgments

I.D. is supported by a stipend from the Helmholtz International Graduate School for Infection Research; L.C.S. and M.M. are supported by the Helmholtz Virtual Institute “Viral Strategies of Immune Evasion” (VH-VI-424).

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Iryna Dekhtiarenko
    • 1
  • Luka Čičin-Šain
    • 2
    Email author
  • Martin Messerle
    • 3
  1. 1.Department of VaccinologyHelmholtz Centre for Infection ResearchBraunschweigGermany
  2. 2.Helmholtz Centre for Infection ResearchBraunschweigGermany
  3. 3.Institute of Virology, Hannover Medical SchoolHannoverGermany

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