A self-recombining bacterial artificial chromosome and its application for analysis of herpesvirus pathogenesis
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Edited by Sankar Adhya, National Institutes of Health, Bethesda, MD, and approved February 15, 2000 (received for review November 17, 1999)
Abstract
A self-recombining bacterial artificial chromosome (BAC) containing the 142-kb pseudorabies virus genome was constructed such that the viral genome is rapidly excised from the BAC vector backbone on delivery into mammalian cells. The recombination is mediated by loxP sites in the plasmid and Cre recombinase encoded within the BAC vector. A synthetic intron inserted in the middle of the cre ORF completely inhibits recombination in Escherichia coli, but is spliced out after delivery of the plasmid into mammalian cells. Recombination is efficient, and pure virus lacking the BAC vector backbone is immediately isolated from transfected mammalian cells without the need of serial passage or plaque purification.
Footnotes
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↵ * To whom reprint requests should be addressed. E-mail: lenquist{at}molbiol.princeton.edu.
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This paper was submitted directly (Track II) to the PNAS office.
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Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.080502497.
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Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.080502497
- Abbreviations:
- BAC,
- bacterial artificial chromosome;
- PRV,
- pseudorabies virus;
- MCMV,
- mouse cytomegalovirus;
- β-gal,
- β-galactosidase;
- GFP,
- green fluorescent protein;
- PK15,
- porcine kidney 15 cells
- Copyright © The National Academy of Sciences
