Transient expression assay in a baculovirus system using firefly luciferase gene as a reporter

Abstract

Transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) in insect cells of Bombyx mori and Spodoptera frugiperda, respectively. DNA sequences encoding luciferase (luc) of the firefly Photinus pyralis was successfully employed in the expression assay as a reporter gene. Recombinant plasmids were constructed containing the luc gene under control of baculovirus-specific or heterologous promoters. Cotransfection of Bombyx mori and Spodoptera frugiperda cells with recombinant plasmids carrying virus-specific promoter sequences and BmNPV and AcNPV DNA, respectively, gave rise to efficient synthesis of luciferase (Luc), while heterologous promoters induced a low level of luc expression. We found that flanking sequences of the AcNPV DNA in the transfer plasmid contained an unknown promoter conferring an efficient luc expression. The activity of this promoter was modulated by the polh promoter sequences. The assay allows one to conduct highly sensitive monitoring of the transient expression of foreign genes from the transfecting plasmids prior to construction of recombinant viruses.

A Low-Cost, Sensitive Reporter System Using Membrane-Tethered Horseradish Peroxidase for Efficient Gene Expression Analysis

“…Firefly luciferase (FLuc) is the most recognized reporter gene. Through DNA engineering, FLuc is often embedded after a promoter of the target gene. − When the promoter is activated through different responses, the downstream genes and FLuc are also transcribed. d -Luc is a substrate of FLuc.…”

Transient expression in insect cells using a recombinant baculovirus synthesising bacteriophage T7 RNA polymerase

“…The generation of plaqued and titred stocks of recombinant viral vectors usually takes 2-3 weeks making the system slow for testing several constructs. Transient assays have been developed based on the introduction into baculovirus infected cells of plasmids containing the foreign gene downstream of an appropriate baculovirus promoter (9,10). In this report we describe an alternative transient expression system for insect cells.…”

Engineering Oncolytic Vaccinia Viruses for Non-Invasive Optical Imaging of Tumors

“…The Rluc substrate (CTZ) is diffusable to many tissues upon tail-vein injection and is non-toxic for mammalian cells [30]. Previously, we reported optical imaging of virus-expressed luciferases and GFP for analyses of gene expression and viral replication in cultured mammalian cells, live insect larvae, and experimental animals [31][32][33][34][35][36][37][38][39]. Noninvasive bioluminescent imaging of luc-labeled murine tumor models has also been shown to correlate with the tumor volume [18] and number of labeled cancer cells injected to animal [40].…”