Baculovirus mediated high-level expression of luciferase in silkworm cells and larvae.
Bombyx mori nuclear polyhedrosis virus (BmNPV)-based baculovirus expression system exploits silkworm larvae as an economical alternative to large-scale cell cultures for production of biomolecules. To generate recombinant BmNPV at high frequencies, we have achieved high efficiency transfection of B. mori cells, BmN, through lipofection. Optimal conditions for lipofection were standardized by quantification of the transient expression level of firefly luciferase (luc) reporter gene under control of an immediate early gene promoter of BmNPV. Lipofection was 50-fold and 100-fold more efficient than the calcium phosphate method for transfecting BmN and Sf9 cells, respectively. Lipofection enabled us to generate a recombinant BmNPV (vBmluc), harboring luc under control of the strong polyhedrin promoter. On infection with vBmluc, luciferase was expressed at very high levels, 170 micrograms/10(6) BmN cells or 13 mg/larva. Expression of luciferase in vBmluc-infected larvae was visualized by luminescence emission instantaneously following luciferin injection generating "glowing silkworms".
Palhan
V B
VB
Indian Institute of Science, Bangalore.
Sumathy
S
S
Gopinathan
K P
KP
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000235:genetics*; D007814:Larva / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins; D008156:Luciferases