Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae.
Bacterial luciferase, derived from a fusion of the luxA and luxB genes of Vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. The coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. These vectors contained the beta-galactosidase indicator gene under control of immediate early (IE1), early (ETL), or very late (P10) promoters and a cloning site for inserting the fused luciferase gene next to the polyhedrin promoter. Recombinant baculoviruses containing the luciferase gene as well as the beta-galactosidase gene could be easily selected when Bluo-gal (beta-galactosidase indicator) was included in the plaque assays. Using cells derived from the fall armyworm (Spodoptera frugiperda), luciferase was strongly expressed very late in infection (48-72 h). The bacterial luciferase assay was sufficiently sensitive that light production could be detected from an extract of a single cell. In addition, live insects, including the cabbage looper (Trichoplusia ni) and saltmarsh caterpillar (Estigmene acrea) were infected by mixing recombinant baculovirus into their diet. Cabbage loopers (with an average wet weight of 223 mg) produced at least 195 micrograms of active luciferase and levels of synthesis peaked between 96-120 h. The results indicate that bacterial luciferase may be used as a reporter of gene expression in insects.
Richardson
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CD
Virology Group, Biotechnology Research Institute, National Research Council of Canada.
Banville
M
M
Lalumière
M
M
Vialard
J
J
Meighen
E A
EA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D007814:Larva / Q000382:microbiology; D008156:Luciferases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009036:Moths / Q000382:microbiology*; D000077183:Occlusion Body Matrix Proteins; D010783:Photometry; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012641:Selection, Genetic; D014162:Transfection; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins; D001616:beta-Galactosidase / Q000235:genetics
D000077183:Occlusion Body Matrix Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D008156:Luciferases; D001616:beta-Galactosidase