Microbiology. Expression of the firefly luciferase gene in vaccinia virus: A highly sensitive gene marker to follow virus dissemination in tissues of infected animals.
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 1667-1671, March 1988 Microbiology
Expression of the firefly luciferase gene in vaccinia virus: A highly sensitive gene marker to follow virus dissemination in tissues of infected animals JOSE F. RODRIGUEZ*, DOLORES RODRIGUEZ*, JUAN-RAMON RODRIGUEZ*, ELEANOR B. MCGOWAN*, AND MARIANO ESTEBAN*t* Departments of *Biochemistry and tMicrobiology and Immunology, State University of New York Health Science Center at Brooklyn, New York, NY 11203
Communicated by Chandler McC. Brooks, November 9, 1987
We have Introduced the firefly luciferase ABSTRACT gene of Photinus pyralis into the vaccinia virus genome. This gene is expressed in a coordinate fashion during virus infection. Luminescence produced by the action of luciferase [Photinus-luciferin:oxygen 4-oxidoreductase(decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was easily detectable in infected cells in culture as well as in cells of tissues of infected mice. The limits of detection were about one infected cell in a background of a million noninfected cells. The luciferase assay was about 1000-fold more sensitive than that of .8galactosidase. Our findings show that the luciferase assay can be conveniently used to follow viral gene expression and virus dissemination both in cell cultures and in tissues of infected animals.
experimental animals. This reporter gene could be effectively used to follow virus replication and levels of gene expression in animals infected with vaccinia virus recombinants.
MATERIALS AND METHODS Virus and Cells. The WR strain of vaccinia virus was
The firefly luciferase gene isolated from a cDNA library from Photinus pyralis has been expressed in bacteria (1), plants (2), and animal cells (3). This gene encodes an enzymatically active polypeptide with an apparent molecular mass of 62 kDa, which is targeted to peroxisomes in both firefly and mammalian cells (4). In the presence of luciferin and ATP, an enzyme-bound luciferyl-adenylate complex is formed, and this is followed by oxidative decarboxylation with production of C02, oxyluciferin, AMP, and light (3). The reaction catalyzed by P. pyralis luciferase (EC 1.13.12.7) emits a yellow-green light at pH 7.5 to 8.5, with the peak emission at 560 nm. This light emission can be measured spectrophotometrically or by exposure to x-ray film (2). Because luciferase assays are more sensitive than those assays for any other reporter gene, it has been proposed that this gene could be a new tool for studying gene expression in both plant and animal cells (2, 3). Vaccinia virus is a 185-kilobase (kb) DNA-containing virus that replicates in the cytoplasm of infected cells. During virus infection, extensive transcription occurs, and a high yield of virus, about 10,000 particles, is produced per cell (5). This virus is now being used as a eukaryotic viral vector with potential for vaccine production. This potential exists because virtually any foreign gene of prokaryotic or eukaryotic origin can be introduced into the viral genome and faithfully expressed during virus infection, and, when recombinant viruses are inoculated in animals, there is activation of both cell-mediated and humoral immune responses to the foreign antigen (6). In this investigation, we have introduced the firefly luciferase gene into the genome of vaccinia virus and have provided direct evidence that recombinants of vaccinia virus expressing luciferase can be conveniently used to follow viral gene expression in a few infected cells. Moreover, luciferase activity can easily be measured in target tissues of
propagated in African green monkey kidney BSC-40 cells and purified as described previously (7). BSC-40 cells were grown in Dulbecco's modified Eagle's medium containing 10% newborn calf serum. Construction of Vaccinia Virus Recombinants. Recombinant viruses were prepared by infecting BSC-40 cells with vaccinia virus [0.01 plaque-forming unit (pfu) per cell] and transfecting them with calcium phosphate-precipitated plasmid DNA (10 pug per 60-mm dish). Cell cultures were harvested 48 hr postinfection, and recombinant viruses were isolated by plaque assay after addition of 5-bromo-4-chloro3-indolyl j3-D-galactoside to the agar overlay (8). Blue plaques were picked up and were plaque purified, and stocks of recombinant viruses were prepared in BSC-40 cells. To confirm the predicted structures of the recombinant virus, DNA from infected cells was examined by restriction endonuclease analysis and DNA hybridization. Measurement of Luciferase and 8-Galactosidase Activities. An instrument described by Charo et al. (9) for measuring the luciferin/luciferase luminescent reaction with ATP was used to measure the level of luciferase in culture cells or in animal tissue extracts. The reactions were carried out in glass cuvettes with mechanical stirring of the contents. The reaction mixture consisted of 450 A.l of buffer containing 25 mM Hepes (pH 7.4), 136 mM NaCI, 1 mM EDTA, 6 mM MgSO4, 0.6% polyethylene glycol 6000, and 20-100 ,.l of cell extract (4.6 x 103 cells is 0.3 Ag of protein) or 75 1.l of tissue extract. The shutter was opened to establish the baseline in the absence of exogenous ATP and was closed. Then, a mixture of 25 ul 0.1 M ATP (pH 6.8) and 10 p.l of 10 mM luciferin (Analytical Luminescence Laboratory, San Diego, CA; dissolved in 10 mM Hepes, pH 7.5/150 mM NaCI/1 mM EDTA) was added to start the reaction, and the shutter was quickly opened (
