11 luciferase imaging of gene expression rhythms 269 11 Real Time Reporting of Circadian Regulated Gene Expression by Luciferase Imaging in Plants and Mammalian Cells By David K Welsh Takato Imaizumi and Steve A Kay Abstract Luciferase enzymes have been used as reporters of circadian rhythms in organisms as diverse as cyanobacteria plants fruit flies and mice This article details methodology for real time reporting of circadian regulated gene expression by imaging of luciferase bioluminescence in plants and mammalian cells Introduction Luciferases are naturally occurring protein enzymes that catalyze emission of light from a substrate luminescence Many structurally heterogeneous forms of luciferase exist in a wide range of species Greer and Szalay 2002 The best known is firefly luciferase which catalyzes the emission of green 560 nm photons from its natural substrate firefly luciferin in the presence of oxygen and ATP Many luciferases have relatively short half lives of just a few hours or even 1 h in some cases Leclerc et al 2000 Yamaguchi et al 2003 making them good reporters of changes in transcriptional rate on a circadian timescale Genes for firefly luciferase and a few others have been cloned and are available commercially Luciferase luminescence is extremely dim often too dim to be seen by the unaided eye in experimental applications Various much brighter fluorescent reporters are available Zhang et al 2002 and two of these have been used successfully to monitor circadian rhythms green fluorescent protein GFP Kuhlman et al 2000 and Cameleon a sensor of cytoplasmic Ca2 Ikeda et al 2003 Luciferase however enjoys two principal advantages over the fluorescent reporters First luciferase does not require exogenous illumination the light detected is emitted from the biological sample itself Fluorescent reporters on the other hand require bombardment with toxic relatively high energy photons so that the reporter can be excited and then re emit lower energy photons Over longterm circadian experiments phototoxicity is a serious concern with fluorescent reporters but a nonissue with luciferase Second background emission of light by biological samples or by luciferase substrate alone is generally METHODS IN ENZYMOLOGY VOL 393 Copyright 2005 Elsevier Inc All rights reserved 0076 6879 05 35 00 270 tracking circadian control of gene activity 11 extremely low whereas autofluorescence background levels can be as high as the fluorescence signal of the reporter itself Billinton and Knight 2001 GFP fluorescence for example spectrally overlaps that of riboflavin vitamin B2 an essential enzyme cofactor found in biological tissues and culture media Zylka and Schnapp 1996 Thus despite its modest photic output luciferase is well suited as an optical reporter of circadian function The first use of a luciferase to monitor circadian function was in Gonyaulax a marine dinoflagellate exhibiting a natural circadian rhythm of bioluminescence Hastings 1989 Subsequently exogenous luciferase genes under control of promoters conferring circadian regulation have been introduced into a wide variety of organisms including cyanobacteria Synechococcus Kondo et al 1993 plants Arabidopsis Millar et al 1992 insects Drosophila Brandes et al 1996 Plautz et al 1997 and rodents Asai et al 2001 Wilsbacher et al 2002 Yamaguchi et al 2003 Yamazaki et al 2000 Yoo et al 2004 These transgenic organisms exhibit robust although quite dim circadian rhythms of bioluminescence useful for a wide range of genetic and biochemical studies of circadian clock mechanisms In mammals luciferase has been used to report circadian rhythms of gene expression in peripheral tissue explants Yamazaki et al 2000 cultured suprachiasmatic nucleus SCN slices Yamaguchi et al 2003 and even in vivo Yamaguchi et al 2001 This article focuses on methodologies used in our laboratory for monitoring circadian output from plant seedlings and mammalian cells Plants Vectors For real time monitoring of circadian rhythms in plants we use Arabidopsis thaliana seedlings transgenic for the firefly luciferase gene under control of a promoter conferring circadian transcription The circadian promoter elements we use routinely are derived from the chlorophyll a b binding protein 2 cab2 gene Millar et al 1992 or the cold circadian rhythm RNA binding 2 ccr2 gene Strayer et al 2000 We use a modified luciferase sequence luc available from Promega which is 10 100 brighter than the native luciferase when expressed in plants Binary vectors developed in our laboratory incorporate luc as well as sequences necessary for delivery and expression of the gene in plants Our standard cab2 and ccr2 reporter lines were created using the respective promoter elements inserted into a promoterless vector pATM DOmega For analysis of cis elements in promoters we use a vector containing a minimal nopaline synthase nos 11 luciferase imaging of gene expression rhythms 271 promoter pATM nos The vectors their sequences and the transgenic plants are available upon request Plant Culture and Transformation Seeds are surface sterilized with bleach and embedded into agar plates made with Murashige Skoog MS salts Caisson Labs MSP001 3 sucrose pH 5 8 in 8 g L agar Plants are grown under sterile conditions in an incubator set at 23 and entrained for 5 10 days to an LD 12 12 lighting cycle 12 h light 12 h dark light is 50 60 mol m2 s cool white fluorescent light Plants are transformed using conventional agrobacterium mediated methods Clough and Bent 1998 and transformants are selected by gentamycin or kanamycin drug resistance in MS agar plates with 75 mg L gentamycin or 50 mg L kanamycin Equipment and Reagents To monitor luciferase activity we use a VIM intensified chargecoupled device CCD camera equipped with an ARGUS 50 photoncounting imaging system Hamamatsu For some experiments we have also used a Night Owl cooled CCD camera EG G Berthold See later for a discussion of state of the art CCD cameras Depending on the desired field of view we use wide angle 17 or 35 mm or standard 50 mm photographic lenses mounted on the camera The light collecting ability of a photographic lens depends on its maximal aperture and is inversely proportional to the square of its f number f so lenses with low f are recommended e g Schneider Kreuznach 17 mm f 0 95 Nikon Nikkor 35 mm f 1 4 or 50 mm f 1 2 A light tight black box is used for imaging and the camera mount should have a light tight single or double O ring seal Light