Use of virion DNA as a cloning vector for the construction of mutant and recombinant herpesviruses

S. Monroe Duboise, Jie Guo, Ronald Charles Desrosiers, Jae U. Jung

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

We have developed improved procedures for the isolation of deletion mutant, point mutant, and recombinant herpesvirus saimiri. These procedures take advantage of the absence of NotI and AscI restriction enzyme sites within the viral genome and use reporter genes for the identification of recombinant viruses. Genes for secreted engineered alkaline phosphatase and green fluorescent protein were placed under simian virus 40 early promoter control and flanked by NotI and AscI restriction sites. When permissive cells were cotransfected with herpesvirus saimiri virion DNA and one of the engineered reporter genes cloned within herpesvirus saimiri sequences, recombinant viruses were readily identified and purified on the basis of expression of the reporter gene. Digestion of recombinant virion DNA with NotI or AscI was used to delete the reporter gene from the recombinant herpesvirus saimiri. Replacement of the reporter gene can be achieved by NotI or AscI digestion of virion DNA and ligation with a terminally matched fragment or, alternatively, by homologous recombination in cotransfected cells. Any gene can, in theory, be cloned directly into the virion DNA when flanked by the appropriate NotI or AscI sites. These procedures should be widely applicable in their general form to most or all herpesviruses that replicate permissively in cultured cells.

Original languageEnglish (US)
Pages (from-to)11389-11394
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number21
DOIs
StatePublished - Oct 15 1996
Externally publishedYes

Fingerprint

Genetic Vectors
Saimiriine Herpesvirus 2
Herpesviridae
Reporter Genes
Virion
DNA
Digestion
Viruses
Simian virus 40
Recombinant DNA
Viral Genome
Homologous Recombination
Green Fluorescent Proteins
Genes
Ligation
Alkaline Phosphatase
Cultured Cells
Enzymes

Keywords

  • γ-2 herpesvirus
  • gene transfer
  • T lymphocyte
  • viral oncogenesis

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Use of virion DNA as a cloning vector for the construction of mutant and recombinant herpesviruses. / Duboise, S. Monroe; Guo, Jie; Desrosiers, Ronald Charles; Jung, Jae U.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 93, No. 21, 15.10.1996, p. 11389-11394.

Research output: Contribution to journalArticle

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