Identification of recombinant baculoviruses using green fluorescent protein as a selectable marker.
A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperda cells resulted in the synthesis of a 30-kDa protein, which was confirmed as GFP by Western blotting and by the emission of green fluorescence when illuminated with longwave UV light (495 or 365 nm). To use GFP as a marker for the selection of recombinant baculoviruses, we prepared a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of lacZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfection to prepare recombinant baculoviruses, the virus DNA was linearized with Bsu361 to improve the recovery of non-parental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with the non-parental phenotype. Plaques were rapidly identified by simply exposing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains.
Wilson
L E
LE
Oxford Brookes University, NERC Institute of Virology and Environmental Microbiology, England, UK.
Wilkinson
N
N
Marlow
S A
SA
Possee
R D
RD
King
L A
LA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D015870:Gene Expression; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008856:Microscopy, Fluorescence; D010641:Phenotype; D011994:Recombinant Proteins / Q000235:genetics; D018411:Spodoptera; D014466:Ultraviolet Rays; D010948:Viral Plaque Assay
D004274:DNA, Recombinant; D004279:DNA, Viral; D005819:Genetic Markers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins