Autoexcision of bacterial artificial chromosome facilitated by terminal repeat-mediated homologous recombination: A novel approach for generating traceless genetic mutants of herpesviruses

Fuchun Zhou, Qiuhua Li, Scott Wong, Shou Jiang Gao

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Infectious bacterial artificial chromosomes (BACs) of herpesviruses are powerful tools for genetic manipulation. However, the presence of BAC vector sequence in the viral genomes often causes genetic and phenotypic alterations. While the excision of the BAC vector cassette can be achieved by homologous recombination between extra duplicate viral sequences or loxP site-mediated recombination, these methods either are inefficient or leave a loxP site mark in the viral genome. Here we describe the use of viral intrinsic repeat sequences, which are commonly present in herpesviral genomes, to excise the BAC vector cassette. Using a newly developed in vitro transposon-based cloning approach, we obtained an infectious BAC of rhesus rhadinovirus (RRV) strain RRV26-95 with the BAC vector cassette inserted in the terminal repeat (TR) region. We showed that the BAC vector cassette was rapidly excised upon reconstitution in cells predominantly through TR-mediated homologous recombination. Genetic and phenotypic analysis showed that the BAC-excised virus was reversed to wild-type RRV. Using this autoexcisable BAC clone, we successfully generated an RRV mutant with a deletion of Orf50, which encodes a replication and transcription activator (RTA) protein. Together, these results illustrate the usefulness of TR for genetic manipulation of herpesviruses when combined with the novel transposon-based cloning approach.

Original languageEnglish (US)
Pages (from-to)2871-2880
Number of pages10
JournalJournal of Virology
Volume84
Issue number6
DOIs
StatePublished - Mar 2010

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Bacterial Artificial Chromosomes
bacterial artificial chromosomes
terminal repeat sequences
Terminal Repeat Sequences
Herpesviridae
Homologous Recombination
homologous recombination
Macacine herpesvirus 5
mutants
Rhadinovirus
Viral Genome
genetic engineering
transposons
genome
Organism Cloning
molecular cloning
Genetic Recombination
transcription (genetics)
Clone Cells
Genome

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

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title = "Autoexcision of bacterial artificial chromosome facilitated by terminal repeat-mediated homologous recombination: A novel approach for generating traceless genetic mutants of herpesviruses",
abstract = "Infectious bacterial artificial chromosomes (BACs) of herpesviruses are powerful tools for genetic manipulation. However, the presence of BAC vector sequence in the viral genomes often causes genetic and phenotypic alterations. While the excision of the BAC vector cassette can be achieved by homologous recombination between extra duplicate viral sequences or loxP site-mediated recombination, these methods either are inefficient or leave a loxP site mark in the viral genome. Here we describe the use of viral intrinsic repeat sequences, which are commonly present in herpesviral genomes, to excise the BAC vector cassette. Using a newly developed in vitro transposon-based cloning approach, we obtained an infectious BAC of rhesus rhadinovirus (RRV) strain RRV26-95 with the BAC vector cassette inserted in the terminal repeat (TR) region. We showed that the BAC vector cassette was rapidly excised upon reconstitution in cells predominantly through TR-mediated homologous recombination. Genetic and phenotypic analysis showed that the BAC-excised virus was reversed to wild-type RRV. Using this autoexcisable BAC clone, we successfully generated an RRV mutant with a deletion of Orf50, which encodes a replication and transcription activator (RTA) protein. Together, these results illustrate the usefulness of TR for genetic manipulation of herpesviruses when combined with the novel transposon-based cloning approach.",
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