Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.
Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location.
Kitts
P A
PA
NERC Institute of Virology and Environmental Microbiology, Oxford, UK.
Ayres
M D
MD
Possee
R D
RD
D000818:Animals; D001426:Bacterial Proteins; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D009036:Moths; D010957:Plasmids; D014162:Transfection
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004279:DNA, Viral; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific; C062194:endodeoxyribonuclease SauI