• Decrease font size
  • Return font size to normal
  • Increase font size
U.S. Department of Health and Human Services

Scientific Publications by FDA Staff

  • Print
  • Share
  • E-mail
-

Search Publications



Fields



Centers











Starting Date


Ending Date


Order by

Entry Details

BMC Biotechnol 2007 May 14;7:22

Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes.

Schmeisser F, Weir JP

Schmeisser F (reprint author), US FDA, Lab DNA Viruses, Ctr Biol Evaluat & Res, Bethesda, MD 20892 USA US FDA, Lab DNA Viruses, Ctr Biol Evaluat & Res, Bethesda, MD 20892 USA

Abstract

BACKGROUND: Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. RESULTS: Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. CONCLUSION: Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.


Category: Journal Article
PubMed ID: #17501993
PubMed Central ID: #PMC1885250
Includes FDA Authors from Scientific Area(s): Biologics
Entry Created: 2011-10-04 Entry Last Modified: 2012-08-29
Feedback
-
-