FLOW CYTOMETRIC ANALYSIS OF BIOLUMINESCENCE EMITTED BY RECOMBINANT BACULOVIRUS-INFECTED INSECT CELLS

Five recombinant baculoviruses, each containing a different insect luc iferase gene encoding a protein with characteristic light emission pro perties, namely, luc GR (546 nm), luc FF (556 nm), luc YG (560 nm), lu c YE (578 nm), and luc OR (593 nm) were constructed. All genes were in serted under the transcriptional control of the polyhedrin gene promot er of the Autographa californica nuclear polyhedrosis virus (AcNPV) an d expressed in Spodoptera frugiperda insect cells during viral infecti on. The biological activity of the different luciferases was character ized by using intact recombinant baculovirus infected cells. Addition of the substrate, D-luciferin, immediately prior to the analysis allow ed monitoring of light emission by flow cytometry. Also, the kinetics of the light emission of lucGR was analyzed with the flow cytometer. T he emission peaks of the infected cells were clearly separated by wave length scanning. Especially, the firefly luciferase (lucFF) had a broa d peak and transient luminescence. The highest maximal intensity value s in vivo were recorded for luc GR and luc YG. SDS-PAGE analysis showe d that the major protein expressed had a molecular weight similar to a uthentic luciferase. Flow cytometry and insect luciferases with clearl y separated emission spectra appear to be of value for sensitive in vi vo analysis of gene promoter activity. (C) 1994 Wiley-Liss, Inc.