A bacmid approach to the genetic manipulation of granuloviruses.
A Cydia pomonella granulovirus (CpGV) bacmid has been constructed, which allows rapid and efficient production of recombinant baculoviruses in Escherichia coli. An 8.6kbp bacterial DNA cassette derived from the AcMNPV Bac-to-Bac system was ligated into a unique PacI 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 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.
Hilton
Sally
S
Warwick HRI, The University of Warwick, Wellesbourne, Warwick CV35 9EF, UK. sally.hilton@warwick.ac.uk
Kemp
Elizabeth
E
Keane
Gary
G
Winstanley
Doreen
D
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000821:virology; D005216:Fat Body / Q000201:enzymology / Q000821:virology; D005814:Genes, Viral / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D025722:Granulovirus / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007814:Larva / Q000201:enzymology / Q000821:virology; D009036:Moths / Q000166:cytology / Q000821:virology; D011994:Recombinant Proteins / Q000235:genetics
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases