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


CONCLUSIONS
Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.
Schmeisser
Falko
F
Laboratory of DNA Viruses, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA. falko.schmeisser@fda.hhs.gov
Weir
Jerry P
JP
D010582:Bacteriophage lambda / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D018139:Simplexvirus / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins