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A bacmid approach to the genetic manipulation of granuloviruses

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Hilton, Sally, Kemp, E. (Elizabeth), Keane, G. (Gary) and Winstanley, Doreen (2008) A bacmid approach to the genetic manipulation of granuloviruses. Journal of Virological Methods, Vol.152 (No.1-2). pp. 56-62. doi:10.1016/j.jviromet.2008.05.015

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Official URL: http://dx.doi.org/10.1016/j.jviromet.2008.05.015

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Abstract

A Cydia pomonella granulovirus (CpGV) bacmid has been constructed, which allows rapid and efficient production of recombinant baculoviruses in Escherichia coli. An 8.6 kbp bacterial DNA cassette derived from the AcMNPV Bac-to-Bac (R) system was ligated into a unique Pacl restriction site within an intergenic region flanking the DNA ligase gene of the CpGV genome. The CpGV bacmids produced in E. coli were transfected into a CpGV-permissive C. pomonella cell line and the transfected cells fed to larvae to amplify the virus. The enhanced green fluorescent protein (EGFP) gene under the constitutive Drosophila heat-shock promoter was transposed into the mini-attTn7 transposition site, using a modified pFASTBAC (TM) donor plasmid, to generate a recombinant CpGV bacmid which caused infected larvae to glow under UV light. Targeted homologous recombination was also achieved in a recombinant proficient E. coli strain (BJ5183). A chloramphenicol acetyl transferase (CAT) gene replaced the cathepsin (v-cath) gene in the bacmid to produce a v-cath-deletion mutant. This is the first published report of a granulovirus bacmid, which will allow easy manipulation of the CpGV genome, enabling future studies on granulovirus genes and biology. (C) 2008 Elsevier B.V. All rights reserved.

Item Type: Journal Article
Subjects: Q Science > QH Natural history > QH426 Genetics
Q Science > QR Microbiology > QR355 Virology
Divisions: Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) > Warwick HRI (2004-2010)
Library of Congress Subject Headings (LCSH): Baculoviruses -- Genetics, Codling moth -- Pathogens, Microbial genetic engineering
Journal or Publication Title: Journal of Virological Methods
Publisher: Elsevier BV
ISSN: 0166-0934
Official Date: September 2008
Dates:
DateEvent
September 2008Published
Volume: Vol.152
Number: No.1-2
Number of Pages: 7
Page Range: pp. 56-62
DOI: 10.1016/j.jviromet.2008.05.015
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Restricted or Subscription Access
Funder: Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)

Data sourced from Thomson Reuters' Web of Knowledge

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