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 Bsu36I to improve the recovery of nonparental
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.