Abstract
We have developed a fast and sensitive system for the in vivo analysis of gene expression in baculovirus infected lepi dopteran insect cells. A recombinant baculovirus containing a luciferase gene from the click beetle, Pyrophorus plagiophthalamus, under transcriptional regulation of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) was used to infect a Spodoptera frugiperda cell line. Recombinant luciferase could be monitored by luminometry in real–time without disruption of the infected cells, allowing detection of synthesis as early as one hour after infection. The range of luminescence measurements was normally over four orders of magnitude, and the kinetics of luciferase synthesis and the levels of light produced in vivo closely correlated with the expression of polyhedrin in AcNPV infected cells when analyzed by SDS PAGE. Additionally, single infected cells could be identified by CCD image analysis and flow cytometry.
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References
Granados, R.R. and Federici, B.A. 1986. The Biology of Baculoviruses, Vol 1. CRC Press, Boca Raton, FL.
Summers, M.D. and Smith, G.E. 1987. A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agricultural Experiment Station No. 1555.
Miller, L.K. 1988. Baculoviruses as gene expression vectors. Ann. Rev. Microbiol. 42: 177–179.
Webb, N.R. and Summers, M.D. 1991. Expression of proteins using recombinant baculoviruses. Technique 4: 173–188.
Valk, J.M. and Keus, R.J.A. 1990. Baculovirus Expression Vector System for Production of Viral Vaccines, p. 91–128. In: Viran Vaccines. Wiley-Liss Inc., NY.
Luckow, V.A. and Summers, M.D. 1988. Trends in the development of baculovirus expression vectors. Bio/Technology 6: 47–55.
Luckow, V.A. and Summers, M.D. 1988. Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vector. Virology 167: 56–71.
Luckow, V.A. and Summers, M.D. 1989. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vector. Virology 170: 31–39.
Seliger, H.H. and McElroy, W.D. 1964. The colors of firefly bioluminescence: enzyme configuration and species specificity. Proc. Natl. Acad. Sci. USA 52: 75–81.
deWet, J.R., Wood, K.V., Helinski, D.R. and DeLuca, M. 1985. Cloning of firefly luciferase cDNA and the expression of active luciferase in Excherichia Coli. Proc. Acad. Natl. Sci. USA 80: 7870–7873.
Ow, D.W., Wood, K.V., DeLuca, M., de Wet, J.R., Helinski, D.R. and Howell, S. 1986. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234: 856–859.
de Wet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R. and Subramani, S. 1987. The firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7: 725–737.
Keller, G.A., Gould, S., DeLuca, M. and Subramani, S. 1987. Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc. Natl. Acad. Sci. USA 82: 3364–3268.
Wood, K.V. 1991. Recent advances and prospects for use of beetle luciferase as genetic reporters, p. 543–546. In: Bioluminescence and Chemiluminescence: Current Status. Proceedings of the VIth International Symposium on Bioluminescence and Chemiluminescence, Cambridge, September P.E. Stanley and L.J. Kricka (Eds.). John Wiley & Sons, Chichester, UK.
Hasnain, S.E. and Nakhai, B. 1990. Expression of the gene encoding firefly luciferase in insect cells using a baculovirus vector. Gene 91: 135–138.
Jha, P.K., Nakhai, B., Sridhar, P., Talwar, G.P. and Hasnain, S.E. 1990. Firefly luciferase, synthesized to very high levels in caterpillars infected with a recombinant baculovirus, can also be used as an efficient reporter enzyme in vivo. FEBS 274: 24–26.
Wood, K.V., Lam, A.Y. and McElroy, W.D. 1989. Introduction to click beetle luciferases and their applications. J. Biolumin. Chemilumin. 4: 289–301.
Wood, K.V., Lam, A.Y., Seliger, H.H. and McElroy, W.D. 1989. Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colors. Science 244: 700–702.
Luckow, V.A. 1991. Cloning and expression of heterologous genes in insect cells with baculovirus vectors, p. 97–153. In: Recombinant DNA Technology and Applications A. Procop, R.K. Bajpai and C.S. Ho (Eds.). McGraw-Hill, Inc, NY.
Vialard, J., Lalumiere, M., Vernet, T., Briedis, D., Alkhatib, G., Henning, D., Levin, D. and Richardson, C. 1990. Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the β-galactosidase gene. J. Virol. 64: 37–50.
Casadaban, M.J. and Cohen, S.N. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138: 179–207.
Maniatis, T., Fritsch, E.F. and Sambrook, J. 1982. Molecular Cloning, a Laboratory Manual. Cold Spring Harbour Laboratory, Cold Spring Harbor, NY.
Laemmli, U.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685.
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Karp, M., Åkerman, K., Lindqvist, C. et al. A Sensitive Model System for In Vivo Monitoring of Baculovirus Gene Expression in Single Infected Insect Cells. Nat Biotechnol 10, 565–569 (1992). https://doi.org/10.1038/nbt0592-565
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DOI: https://doi.org/10.1038/nbt0592-565
