A cellular promoter-based expression cassette for generating recombinant baculoviruses directing rapid expression of passenger genes in infected insects.
We have developed an expression cassette which allows the generation of recombinant baculoviruses that can express passenger genes under the control of a constitutive cellular promoter derived from the cytoplasmic actin gene of the silkmoth Bombyx mori. Silkmoth tissue culture cells which were infected with a recombinant B. mori nuclear polyhedrosis virus (BmNPV) containing the gene-encoding chloramphenicol acetyltransferase (CAT) under the control of this expression cassette expressed significant CAT activity beginning 5 hr postinfection (p.i.). Cells infected with a recombinant BmNPV containing the cat gene under the control of the polyhedrin gene promoter did not express CAT activity until 20 hr p.i. Silkworm larvae were also infected with the two recombinant viruses by hemocelic injections and all larval tissues examined were found to express the cat gene. While significant actin-cassette-driven CAT expression in vivo was first seen at 24 hr p.i., expression from the polyhedrin promoter was not seen until 48 hr p.i. By 60 hr p.i., tissues of larvae infected with the recombinant virus expressing cat under polyhedrin promoter control were found to exhibit sixfold higher CAT activity than those infected with recombinant virus expressing the cat gene under the control of the actin promoter. The 24-hr temporal advantage in expression of a passenger gene in infected larvae indicates that the actin-promoter-based expression cassette or other analogous cellular promoter-based cassettes could be used for generating recombinant baculovirus insecticides which could incapacitate pest insects more quickly than viruses employing the polyhedrin or other late viral promoters for expressing insect-incapacitating proteins.
Johnson
R
R
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
Meidinger
R G
RG
Iatrou
K
K
D000199:Actins / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D012831:Bombyx; D002478:Cells, Cultured; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D015183:Restriction Mapping
D000199:Actins; D011993:Recombinant Fusion Proteins