Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae.
The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order + 35 > + 1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at + 35 nt of the polh.
Sriram
S
S
Microbiology and Cell Biology Department, Indian Institute of Science, Bangalore.
Palhan
V B
VB
Gopinathan
K P
KP
D000818:Animals; D012831:Bombyx / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D017930:Genes, Reporter; D005813:Genes, Synthetic; D007814:Larva / Q000166:cytology / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic
D008156:Luciferases