imaging of light emission from the expression of luciferases in living cells and organisms
TRANSCRIPT
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Received 13 August 2001; accepted 16 October 2001
ABSTRACT: Luciferases are enzymes that emit light in the presence of oxygen and a substrate (luciferin) and which have been usedfor real-time, low-light imaging of gene expression in cell cultures, individual cells, whole organisms, and transgenic organisms. Suchluciferin–luciferase systems include, among others, the bacterial lux genes of terrestrial Photorhabdus luminescens and marine Vibrioharveyi bacteria, as well as eukaryotic luciferase luc and ruc genes from firefly species (Photinus) and the sea panzy (Renillareniformis), respectively. In various vectors and in fusion constructs with other gene products such as green fluorescence protein(GFP; from the jellyfish Aequorea), luciferases have served as reporters in a number of promoter search and targeted gene expressionexperiments over the last two decades. Luciferase imaging has also been used to trace bacterial and viral infection in vivo and tovisualize the proliferation of tumour cells in animal models. Copyright � 2002 John Wiley & Sons, Ltd.
KEYWORDS: luciferases; gene expression; low-light imaging; luciferase expression constructs
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From time immemorial, seamen and fishermen haveobserved ‘lights’ on the water. In the nineteenth century itwas realized that the most frequent cause of suchluminous oceanic phenomena are minute marine organ-isms emitting light—bioluminescence. About 35 yearsago, various luciferases began to be characterized (1, 2)which, in their many forms, in the presence of a substrate,a luciferin, emit light in the visible range underphysiological conditions. Some eukaryotic organisms,such as the firefly (Photinus), have their own luciferin–luciferase light-emitting systems. Many marine organ-isms, however, such as mid-depth fishes and invertebratessuch as molluscs, emit light because of symbioses withluciferase-producing bacteria occurring in highly specia-lized light organs. These luminescent bacteria includetaxa such as Photobacterium phosphoreum, P. leiognathi,Vibrio logei, V. harveyi and V. fischeri.
It is to be expected that a costly characteristic likebiological production of light would be retained only ifluminescent visualizing were advantageous. Biolumines-cence is used as a disguise for fleeing prey, for ventrallight emission to efface an organism’s shadow and render
it invisible from below (3, 4), for luring prey (ceratioidfish), for signalling for courtship and mating, and instress-induced light emission (bioluminescent plankton).One could argue that ever since such metazoanbioluminescent bacteria symbioses and other biolumi-nescent organisms appeared in the oceans with theirunique light emission systems, there has been in vivoluminescent ‘imaging’ or visualization.
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Marine bioluminescence may be considered one of themost widespread forms of communication on the planet.Organisms emit light that other organisms detect or‘visualize’ and to which they give some behavioralresponse (5). Behavior based on natural bioluminescenceimaging may be classified under three general headings (5):offence (luring, baiting); defence (startle, camouflage); andcommunication (courtship and mating). Some striking usesof natural bioluminescent ‘visualization’ include thefollowing: some squids with bacterial symbionts useshadow-effacing, or modulation of their ventrally-emittedlight, to match ambient sunlight or moonlight; crustaceans,similar to fireflies, may use a repetitive mating ‘Morsecode’ of blinks; some jellyfishes deposit an adhesive glowupon contact with predators, leaving the predator visibleand vulnerable; some squids flee, leaving a luminescentcloud of ‘ink’ in a predator’s face; some dragonfishes(Malacosteidae) emit blue-green light, but also emit a‘night-vision’ long-wavelength red light by which they can
Luminescence 2002;17:43–74DOI: 10.1002/bio.676
*Correspondence to: A. A. Szalay, Department of Biochemistry,School of Medicine and Department of Natural Sciences—BiologySection; Loma Linda University, Loma Linda, CA 92354, USA.Email: [email protected]
Contract/grant sponsor: Dept. Natural Sciences, Loma Linda Uni-versity, CA, USA.Contract/grant sponsor: Basic Science Research Grant from LomaLinda University, USA.
Copyright 2002 John Wiley & Sons, Ltd.
REVIEW
detect prey (using reverse fluorescence energy transfer)without their prey seeing them (6–12).
The purpose here is to review the representativescientific imaging applications to which these naturallyoccurring visible light bioluminescent systems, the genesencoding the proteins and their modifications have beenput. However, we first present an overview of theluciferin–luciferase light emission systems.
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Luciferase is a generic name because none of the majorluciferases share sequence homology with each other (5).Luciferases occur in bacteria, fungi, dinoflagellates,radiolarians and about 17 metazoan phyla and 700genera, mostly marine (5, 12, 13). These include Anne-lida (segmented worms), Chordata (some elasmobran-chiomorphs or sharks, many teleosts or bony fishes),Cnidaria (jellyfishes, anthozoans such as the sea pansy,Renilla), Chaetognaths (one species of arrow-worm),Crustacea (many, including ostracods and euphausiidshrimps or krill), Ctenophora (comb jellies), Echinoder-mata (sea stars, brittle stars), hemichordate worms,Insecta (fireflies, click beetles), Mollusca (squids,octopods, nudibranchs), Nemertean worms (one species),Pycnogonids (sea spiders), Urochordata (larvaceans,pyrosomes, and one tunicate), millipedes and centipedes(12). Phylogenetic analyses suggest that luciferin–luciferase systems have had more than 30 independentorigins (5, 14–16).
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Bioluminescence is a chemiluminescent reaction be-tween at least two molecules produced under physio-logical conditions within or in association with anorganism. The substrate molecule reacted upon, whichemits light in such a reaction, is called a luciferin.Luciferases are a wide range of enzymes that catalyse theoxidation of substrate luciferins to yield non-reactiveoxyluciferins and the release of photons of light (17–21).As luciferin substrates are used, they must be replenished,which usually occurs through diet. Some luciferinsrequire the presence of a co-factor to undergo oxidation,such as FMNH2
�, Ca2� or ATP (22). Complexes thatcontain a luciferase, a luciferin, and generally requiringO2 are also called photoproteins (12).
Although luciferin–luciferase bioluminescence isfound in hundreds of taxa across many phyla, there arefive basic luciferin–luciferase system (12):
� Bacterial luciferin is a reduced riboflavin phos-phate (FMNH2) that is oxidized by a luciferase in
association with a long-chain aldehyde and anoxygen molecule. It is found in luminescentbacteria, certain fish, pyrosomes, and in somesquids (e.g. Euprymna).
� Dinoflagellate luciferin resembles, and may bederived from, the porphyrin of chlorophyll. In thedinoflagellate Gonycaulax, this luciferin is con-formationally shielded from luciferase at the basicpH of 8 but becomes free and accessible tooxidation near the more acidic pH of 6. Amodification of this luciferin occurs in a herbivor-ous euphausiid shrimp, where it is apparentlyacquired by ingestion.
� Another luciferin, from the marine ostracod Var-gula, is called vargulin. It also seems to be acquiredby ingestion. It is also found in some fish species.
� Coelenterazine is the most widely known luciferin.It occurs in cnidarians, copepods, chaetognaths,ctenophores, decapod shrimps, mysid shrimps,radiolarians, and some fish taxa. Coelenterateluciferase activity is controlled by the concentra-tion of Ca2� and shares homology with thecalcium-binding protein calmodulin (5).
� Firefly luciferin (a benzothiazole) is found exclu-sively in fireflies (Photinus or Luciola). It has theunique property of requiring ATP as a co-factor toconvert it to an active luciferin (5). It was realizedearly that firefly luciferin–luciferase could be usedto determine the presence of ATP (23). This hasbecome a standard ATP assay. For one example,since nickel alloys have been shown to have anadverse effect on respiratory metabolism in eu-karyotic cell lines, the firefly luciferin–luciferasesystem has been used to document depressed levelsof ATP in cells exposed to the alloys (24).
The mechanisms of bioluminescence utilized byamphipods, bivalves, earthworms, fresh-water limpets,fungus gnats, larvaceans, nemertean worms, polychaeteworms and tunicates are currently unknown. Luciferin–luciferase bioluminescence systems are multiformphenomena and polyphyletic in origin.
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Science has entered into the field of bioluminescentvisualization in far more recent times. In the last fewdecades, many luciferase genes have been isolated,sequenced at least in part, and used to build DNAvectors. In Table 1 we summarize the DNA fragmentsand cDNAs that encode the different luciferases sig-nificant in scientific imaging.
The luciferases most commonly used in experimentalbioluminescent imaging applications include the bacterial
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
44 REVIEW L. F. Greer and A. A. Szalay
Tab
le1.
Asu
mm
ary
ofkn
own
luci
fera
sege
nes,
cDN
As,
and
prot
eins
.Am
ong
thes
ear
eth
epr
okar
yoti
clu
cife
rase
s(L
ux),
euka
ryot
iclu
cife
rase
s(L
uc,R
ucan
dth
eir
regu
lato
rypr
otei
ns)
both
ofw
hich
are
com
mon
lyus
edin
imag
ing
oflu
cife
rase
expr
essi
onin
livin
gce
lls,t
issu
es,a
ndor
gani
sms
Tax
aG
ene
–cD
NA
(siz
ein
bp)
Prot
ein
prod
uct
(siz
ein
num
ber
ofam
ino
acid
s)G
enB
ank
acce
ssio
nno
.(D
NA
and
amin
oac
id)
Ref
eren
ce
Vib
rio
harv
eyi
luxA
,106
7bp
�su
buni
t,35
5aa
M10
961
AA
A88
685
32
Vib
rio
harv
eyi
luxB
,947
bp�
subu
nit,
324
aaM
1096
1.1
AA
A88
686
223
Vib
rio
harv
eyi
luxE
,113
6bp
acyl
-pro
tein
synt
heta
se,
378
aaM
2881
5.1
AA
A27
531
223
Vib
rio
fisch
eri
luxA
,106
4bp
alka
nal
mon
o-ox
ygen
ase�-
chai
n,35
4aa
X06
758
CA
A29
931
224
Vib
rio
fisch
eri
luxB
,980
bpal
kana
lm
ono-
oxyg
enas
e�-
chai
n,32
6aa
X06
797
CA
A29
932
224
Vib
rio
fisch
eri
Lux
RIC
DA
BE
Gop
eron
�lu
xR,7
52bp
�lu
xI,5
81bp
�lu
xC,1
439
bp�
luxD
,923
bp�
luxA
,107
7bp
�lu
xB,9
93bp
�lu
xE,1
136
bp
�lu
xG,7
22bp
�re
gula
tory
prot
ein
Lux
R,2
50aa
�au
toin
duce
rsy
nthe
sis
prot
ein
Lux
I,19
3aa
�ac
yl-C
oAre
duct
ase
Lux
C,4
79aa
�ac
yltr
ansf
eras
eL
uxD
,307
aa�
alka
nal
mon
o-ox
ygen
ase�-
chai
nL
uxA
,354
aa�
alka
nal
mon
o-ox
ygen
ase�-
chai
nL
uxB
,326
aa�
long
chai
nfa
ttyac
idlu
cife
rin
com
pone
ntlig
ase
Lux
E,3
78aa
�pr
obab
lefla
vin
redu
ctas
eL
uxG
,236
aa
AF1
7010
4�
AA
D48
473
�A
AD
4847
4�
AF1
7010
4�
AA
D48
476
�A
AD
4847
7�
AA
D48
478
�A
AD
4847
9
�A
AD
4848
0
Kni
ght
T,P
apad
akis
N.
Vib
rio
fisch
eri
lux
oper
onSa
lI
dige
st(u
npub
lishe
d—di
rect
subm
issi
onto
Gen
Ban
k,19
99)
Pho
torh
abdu
slu
min
esce
ns=
Xen
orha
bdus
;si
nce
1999
redu
ced
tosy
nony
my
(225
)
Lux
CD
AB
Eop
eron
�lu
xC,1
442
bp�
luxD
,923
bp�
luxA
,108
8bp
�lu
xB,9
74bp
�lu
xE,3
47bp
�fa
ttyac
idre
duct
ase
Lux
C,4
80aa
�ac
yltr
ansf
eras
eL
uxD
,307
aa�
alka
nal
mon
o-ox
ygen
ase�-
chai
nL
uxA
,362
aa�
alka
nal
mon
o-ox
ygen
ase�-
chai
nL
uxB
,324
aa�
acyl
-pro
tein
synt
heta
seL
uxE
,116
aa
M62
917
�A
AA
6356
3�
AA
A63
564
�A
AA
6356
5�
AA
A63
566
�A
AA
6356
7
34
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
Imaging of light emission from luciferase expression REVIEW 45
Tab
le1.
Con
tinu
ed
Tax
aG
ene
–cD
NA
(siz
ein
bp)
Prot
ein
prod
uct
(siz
ein
num
ber
ofam
ino
acid
s)G
enB
ank
acce
ssio
nno
.(D
NA
and
amin
oac
id)
Ref
eren
ce
Pho
tinu
spy
rali
slu
c,23
87bp
Luc
ifer
ase,
550
aaM
1507
7A
AA
2979
522
6
Luc
iola
cruc
iata
luc,
1985
bpL
ucif
eras
e,54
8aa
M26
194
AA
A29
135
227
Var
gula
hilg
endo
rfii
(sea
firefl
y)�
vuc,
1834
bp�
vuc
mR
NA
,181
8bp
Var
gulin
,61
1aa
Var
gulin
,55
5aa
E02
749
M25
666
AA
A30
332
228 39
Aeq
uore
avi
ctor
iaae
q1,6
72(5
90)
bpae
qpre
c,56
8bp
aeq2
,531
bpae
q3,5
31bp
aq44
0,92
5bp
aqua
,587
bp
Aeq
uori
n1;
calc
ium
-bin
ding
prot
ein,
196
aaA
equo
rin
prec
urso
r,�1
89aa
Aeq
uori
n2,
177
aaA
equo
rin
3,17
7aa
Aeq
uori
n,ca
lciu
mbi
ndin
g-
196
aaL
umin
esce
ntpr
otei
nA
qual
ine,
�196
M16
103
AA
A27
719
M11
394
AA
A27
716
M16
104
AA
A27
717
M16
105
AA
A27
718
L29
571
AA
A27
720
E02
319
229
230
231
Opl
opho
rus
grac
ilor
ostr
islu
c,59
0bp
luc,
1079
bpO
plop
hori
n,ox
ygen
ase,
imid
azop
yraz
inon
elu
cife
rase
,19
6aa
Opl
opho
rin,
oxyg
enas
e,im
idaz
opyr
azin
one
luci
fera
se�3
59aa
AB
0302
46B
AB
1377
6A
B03
0245
BA
B13
775
52
Ren
illa
mue
ller
iru
c,12
08bp
Ruc
,311
aaA
Y01
5988
AA
G54
094
Szen
t-G
yorg
yiC
S,B
ryan
BJ.
cDN
Aen
codi
ngR
enil
lam
uell
eri
luci
fera
se(u
npub
lishe
dm
anus
crip
t).
Ren
illa
reni
form
isru
c,11
96bp
Ruc
,oxy
gena
se,3
11aa
M63
501
AA
A29
804
59
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
46 REVIEW L. F. Greer and A. A. Szalay
luciferases (lux) from the marine genera Photobacteriumand Vibrio, firefly luciferase (Photinus), aequorin (lucifer-ase from the jellyfish Aequorea), vargulin (luciferase fromthe marine ostracod Vargula), oplophoran luciferase(deep-sea shrimp Oplophorus) and Renilla luciferase(anthozoan sea pansy, Renilla reniformis).
� Bacterial luciferase. Bacterial luciferase proteinswere purified and isolated from the light organs ofmid-depth fishes in the ocean (25, 26). It wasknown early that the catalytic site was on the �subunit (27). Belas et al. (1982) isolated andexpressed luciferase genes from Vibrio harveyi inE. coli (28). Olsson et al. (1988) characterized theactivity of the LuxA subunit of Vibrio harveyiluciferase by visualizing various luxA and luxBtruncations, as well as a luxAB fusion expressed inE. coli (29). Olsson et al. (1989) furthermore mademonomeric luxAB fusions and expressed them alsoin E. coli (30). The Vibrio harveyi luxA and luxBcDNAs were cloned and sequenced in the mid-1980s (31–33). The luxCDABE operon from theterrestrial bacterium Photorhabdus luminescenswas cloned and sequenced and its product, lucifer-ase, was characterized and published in 1991 (34).
� Firefly luciferase. The active sites and properties offirefly luciferase (Photinus) began to be character-ized about 35 years ago (35–37). Firefly luciferasewas purified and characterized in 1978 (19). ThecDNA encoding the luciferase (Luc) from thefirefly Photinus pyralis was cloned and expressedin E. coli by De Wet et al. (1985) (38).
� Vargulin. A cDNA for the luciferase gene from themarine ostracod Vargula hilgendorfii was cloned,sequenced and expressed in mammalian cells byThompson et al. (1989) (39). They also discoveredthat Vargula luciferase expression requires only itssubstrate and molecular oxygen (but no co-fac-tors), thus making it potentially more useful formammalian expression systems (40). The activityof Vargula luciferase is not dependent on apyrazine structure, as has been demonstrated bycross-reaction experiments with the Oplophorusluciferin (41).
� Aequorin. The aequorin protein was first extractedfrom the hydromedusa Aequorea, purified andcharacterized in part by Shimomura et al. (1962)(42). In 1975, Shimomura and Johnson describedwhat was known about the mechanisms of variouscoelenterate luciferins, including aequorin (22).Ward and Cormier (1975) reported the isolation ofvarious Renilla-type luciferins, including aequorin(43). A few years later, it was discovered thatRenilla luciferin analogues were catalysed byluciferase to excited energy states to transferenergy to a green fluorescence protein or GFP
(44). Ward and Cormier (1979) characterized theRenilla green fluorescence protein (RGFP) andshowed that a natural energy transfer was occur-ring from the isolated Renilla luciferase (Ruc)bioluminescence to RGFP (45). In 1985, the cDNAfor aequorin was cloned, sequenced and expressedin heterologous systems (46, 47). The aequoringene from the jellyfish Aequorea victoria wascloned in 1990 (48). It is now known that manycnidarians have GFPs that serve as energy-transferacceptors fluorescing in response to excited oxy-luciferin–luciferase complexes or to a Ca2�-acti-vated phosphoprotein. The cDNA encoding theGFP of Aequorea victoria has also been cloned andsequenced (49).
� Oplophorus luciferase. The general reaction mech-anisms and properties of the luciferin–luciferasesystem of the deep-sea shrimp Oplophorus graci-lorostris were reported by Shimomura et al. (1978)(50). An empirical formula and structure has beensuggested for Oplophorus luciferin using spectro-scopy and cross-reaction with the luciferase of theostracod Vargula hilgendorfii (40). By 1997,Oplophorus luciferase was known to have a moreintense light emission than either Renilla luciferaseor the recombinant aequorin. However the Oplo-phorus luciferase cDNA, not yet cloned, could notbe used as a reporter gene (51). Recently, Inouye etal. (2000) succeeded in cloning the Oplophorusluciferase cDNA (52).
� Renilla luciferase. In 1966, Hori and Cormierdescribed some of the properties and a hypotheticalpartial structure for the Renilla reniformis lucifer-ase protein (Ruc) (53). Kreis and Cormier (1967)showed that light could inhibit the activity of Ruc(54). The isolation of Ruc was first done andfurther properties elucidated by Karkhanis andCormier (1971) (55). DeLuca et al. (1971) demon-strated that the Renilla bioluminescent systeminvolves the oxidative production of CO2 (56). Itwas further shown that Ca2� triggered a luciferinbinding protein, thus inducing the Ruc system (57).Ruc was first purified and characterized byMatthews et al. (1977) (58). The cDNA of rucwas isolated and later expressed in E. coli byLorenz et al. (1991) (59). The ruc cDNA was alsoexpressed in a number of transgenic plant tissues(60). In 1996, Lorenz et al. expressed Ruc insimian COS-7 cells and in murine C5 cells (61).
In retrospect, it might be noted that since theirdiscovery, Luc (Photinus), aequorin (Aequorea) andGFP have been used in a multitude of successfulexperiments. In combination the three have even beenuseful in assaying or imaging the spatial–temporalconcentrations of Ca2� (62). Combinations of multiple
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
Imaging of light emission from luciferase expression REVIEW 47
Tab
le2.
Asu
mm
ary
ofse
lect
edlu
cife
rase
cons
truc
tsan
dve
ctor
sus
eful
for
imag
ing.
(Con
stru
ct/v
ecto
rno
men
clat
ure
not
stan
dard
ized
inth
elit
erat
ure)
.
Con
stru
ct/v
ecto
rL
ucif
eras
ege
nes
orcD
NA
sPr
omot
ers/
enha
ncer
sO
rgan
ism
/cel
lsSu
bstr
ate
requ
irem
ent
Imag
ing
appl
icat
ion
and
refe
renc
e
pB10
1;pB
102;
pB10
5;pB
110;
pB12
3;pB
128;
pGM
C12
luxA
,lux
B(V
ibri
oha
rvey
i)Ph
age�
prom
oter
sP L
and
P RE
sche
rich
iaco
liD
ecan
alE
xpre
ssio
nof
luxA
,lux
Bin
E.
coli
(28)
—fir
sttr
ansg
enic
expr
essi
onof
lux
Tra
nspo
son
min
i-M
ulux
luxE
,B,A
,D,C
(Vib
rio
para
haem
o-ly
ticu
s)la
cpr
omot
erE
.co
liN
one
Vis
ualiz
atio
nof
Vib
rio
lux
gene
sin
E.
coli
(26)
pFIT
001;
pPA
LE
001
luxA
,lux
B(V
.ha
rvey
i)A
nti-
Tet
(P1)
,nif
D,n
ifH
prom
oter
sE
.co
li;
Bra
dyrh
izob
ium
japo
nicu
min
Gly
cine
max
Dec
anal
Vis
ualiz
atio
nof
N-fi
xatio
nin
soyb
ean
nodu
les
via
Bra
dyrh
izob
ium
japo
nicu
m(2
10)
Agr
obac
teri
umbi
nary
vect
orlu
xA&
B(V
.ha
rvey
i)T
Lpr
omot
erD
aucu
sca
rota
;N
icot
iana
taba
cum
Dec
anal
Vis
ualiz
atio
nof
tissu
e-sp
ecifi
cch
imae
ric
lux
expr
essi
on(1
69)
pDO
432;
pDO
435;
pDO
446;
pDO
445
luc
(Pho
tinu
s)C
aMV
35S
RN
Apr
omot
erN
icot
iana
taba
cum
Pho
tinu
slu
cife
rin
and
AT
Pin
solu
tion
(top
ical
lyde
liver
ed)
Vis
ualiz
atio
nof
luc
expr
essi
onin
toba
cco
plan
ts(1
68)
pPC
V70
1lux
A&
Blu
xA;
luxB
(V.
harv
eyi)
TR-D
NA
P1an
dT
R-D
NA
P2
mas
prom
oter
sN
icot
iana
taba
cum
;D
aucu
sca
rota
via
Agr
obac
teri
umtu
mef
acie
ns-
med
iate
dge
nede
liver
y
Dec
anal
(inj
ecte
d)D
ecan
al�
FMN
H2
Ass
embl
yan
dex
pres
sion
offu
nctio
nal
luxA
and
Bge
nes
inpl
ants
(170
)
pRSV
Llu
cbu
tim
aged
byim
mun
o-flu
resc
ence
Prom
oter
inR
ous
Sarc
oma
Vir
uslo
ngte
rmin
alre
peat
(RSV
LT
R)
Mon
key
kidn
eyce
lls(C
V-1
)N
AL
ucif
eras
epe
roxi
som
allo
caliz
atio
nvi
sual
-iz
edby
imm
unofl
uore
scen
ce(1
09)
pMR
P1;
pMR
D2
luxA
Bfu
sion
P1
prom
oter
Soyb
ean
(Gly
cine
max
)D
ecan
alSu
cces
sful
imag
ing
ofL
uxA
Bfu
sion
expr
essi
onin
soyb
ean
root
nodu
les
usin
gph
otog
raph
icfil
man
dlo
w-l
ight
inte
nsifi
edvi
deo
mic
rosc
opy
(211
)pS
CL
UC
→,p
SCL
UC��
,rV
V-l
uclu
c7.
5kD
avi
ral
prom
oter
,Vac
cini
atk
gene
frag
men
tsB
SC-4
0ce
lls(A
fric
angr
een
mon
key
kidn
eyce
lls)
Fire
flylu
cife
rin
Film
imag
ing
ofre
com
bina
ntva
ccin
iain
fect
ion
ofm
onke
yce
lls(2
12)
pLX
vect
orse
ries
luxA
;lu
xB;
luxA
B(l
uxF
)T
7pr
omot
erB
E21
(DE
3)ce
llsD
ecan
alIm
agin
gof
vari
ous
lux
trun
cati
ons
and
Lux
Ffu
sion
ince
lls(2
9)pR
S110
5lu
xA,l
uxB
(V.
harv
eyi)
End
oH,b
ldA
(leu
tRN
A),
Whi
G(u
ncha
ract
eriz
ed),
SapA
(all
Stre
ptom
yces
prom
oter
s)
Stre
ptom
yces
coel
icol
orD
ecan
alV
isua
lizin
glu
xA&
Bex
pres
sion
inSt
rept
omyc
es(9
1)
pLX
,pIC
LX
,pC
V70
2an
dp3
5Slu
xve
ctor
seri
eslu
xAB
;lu
xBA
fusi
ons
T7
gene
10an
dC
aMV
35S
prom
oter
E.
coli
;to
bacc
oca
lliD
ecan
alSh
owed
that
aL
uxA
Bfu
sion
ism
uch
mor
eac
tive
than
Lux
BA
(30)
pLX
vect
orse
ries
luxA
BT
7ge
ne10
prom
oter
E.
coli
Dec
anal
Bac
teri
allu
cife
rase
��fu
sion
func
tiona
las
am
onom
er(6
5)pM
W41
luc
CM
Vin
term
edia
te–e
arly
enha
ncer
/pr
omot
er;
tran
s-ac
tiva
ted
byH
IV-1
Tat
prot
ein
CO
S-7
cells
Luc
ifer
in(fi
refly
)an
dde
riva
tive
s:et
hoxy
viny
les
ter,
2-hy
drox
yeth
yles
ter,
3-hy
drox
y-n-
prop
yles
ter,
ethy
les
ter
Vis
ualiz
atio
nof
Luc
expr
essi
onin
indi
vidu
alm
amm
alia
nce
lls(1
12)
pSV
2-vl
luc
(Var
gula
luci
fera
se)
SV40
prom
oter
CH
Oce
llsL
ucif
erin
Vis
ualiz
atio
nof
luc
secr
etio
nin
CH
Oce
llsre
altim
eus
ing
anim
age-
inte
nsif
ying
tech
niqu
e(1
14)
pPC
VG
Lux
Aan
dB
luxA
and
BPr
omot
erle
sslu
xApC
aMV
35S
RN
A(l
uxB
),i.e
.pr
omot
erse
arch
vect
oras
say
Tob
acco
Dec
anal
Imag
ing
ofco
nstit
utiv
ean
dor
gan-
spec
ific
Lux
expr
essi
onin
tran
sgen
icto
bacc
o(1
72)
pAM
1224
;ps
bAI:
:luxA
Bco
nstr
uct
luxA
B(V
.ha
rvey
i)lu
xAB
prom
oter
less
,(to
bein
sert
eddo
wns
trea
mof
cyan
obac
teri
alpr
o-m
oter
);ps
bAI,
psbA
III
and
purF
prom
oter
s
Syne
choc
occu
ssp
.,ve
ctor
edby
conj
ugat
ion
with
E.
coli
Dec
anal
Coo
led-
CC
Dde
tect
ion
and
docu
men
tatio
nof
circ
adia
nrh
ythm
sin
cyan
obac
teri
a(9
8–10
0)
pPC
VG
luxA
and
Blu
xAan
dB
(V.
harv
eyi)
T-D
NA
prom
oter
sear
chve
ctor
;lu
xApr
omot
erle
ss,b
uten
hanc
edby
CaM
V35
Spr
omot
erin
fron
tof
luxB
Agr
obac
teri
umtu
mef
acie
nsT
-DN
A-
med
iate
dtr
ansf
erof
lux
into
tran
s-ge
nic
Nic
otia
nata
bacu
mcv
.SR
1(e
spec
ially
root
stem
,lea
fan
dflo
wer
tissu
es)
Dec
anal
Imag
ing
used
tolo
cate
deve
lopm
enta
llyre
gula
ted
prom
oter
sin
toba
cco
(96)
pWH
1520
–xyl
A–l
uxF
(Bac
illu
sG
ram
posi
tive)
expr
essi
onve
ctor
luxA
B(l
uxF
;V
.ha
rvey
i)X
ylos
e-in
duci
ble
prom
oter
–lux
Ffu
sion
Xyl
A–l
uxF
-tra
nsfo
rmed
B.
thur
ingi
en-
sis
inje
cted
into
Man
duca
sext
aar
vae
(6th
inst
arof
the
toba
cco
horn
wor
m)
Dec
anal
Use
ofph
oton
-cou
ntin
gto
visu
aliz
e(1
3):
�In
duci
ble
luxF
expr
essi
onfr
omB
.th
urin
-gi
ensi
san
dB
.m
egat
eriu
min
fect
ion
inle
-pi
dopt
eran
inse
ctla
rvae
and
envi
ronm
ent
(and
oflu
xFin
E.
coli
)
������������ �
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
48 REVIEW L. F. Greer and A. A. Szalay
Tab
le2.
Con
tinu
ed
Con
stru
ct/v
ecto
rL
ucif
eras
ege
nes
orcD
NA
sPr
omot
ers/
enha
ncer
sO
rgan
ism
/cel
lsSu
bstr
ate
requ
irem
ent
Imag
ing
appl
icat
ion
and
refe
renc
e
Rec
ombi
nant
AcN
PV–l
uc(A
uto-
grap
hica
cali
forn
ica
nucl
ear
poly
-he
dros
isvi
rus
with
Pluc
)
luxF
(V.
harv
eyi)
;Pl
ucA
rabi
dops
isph
enyl
alan
ine
amm
onia
lyas
e(P
AL
)pr
omot
er–l
uxF
gene
fusi
on;
AcN
PVpo
lyhe
dron
late
prom
oter
(pN
I101
)–lu
c
Rec
ombi
nant
AcN
PV–l
uc-i
nfec
ted
Tri
chop
lusi
ani
(386
)ce
llsD
ecan
al;
luci
feri
n(fi
refly
)U
seof
phot
on-c
ount
ing
imag
ing
tovi
sual
ize
(93)
:�
AcN
PV–l
ucex
pres
sing
plaq
ues
inT
.ni
cells
pPC
VG
luxA
and
Blu
xAan
dlu
xB(V
.ha
rvey
i)lu
xApr
omot
erle
ss,b
uten
hanc
edby
CaM
V35
Spr
omot
erin
fron
tof
luxB
(i.e
.pro
mot
erse
arch
vect
or)
Agr
obac
teri
umtu
mef
acie
nsT
-DN
A-
med
iate
dtr
ansf
erof
lux
into
tran
s-ge
nic
Nic
otia
nata
bacu
mcv
.SR
1fo
rex
pres
sion
inre
spon
seto
vir
and
avir
infe
ctio
nby
Pse
udom
onas
syri
ngae
Dec
anal
Use
ofph
oton
-cou
ntin
gim
agin
gto
visu
aliz
e(9
3):
�Pr
omot
erse
arch
expr
essi
onof
Lux
Aan
dL
uxB
inN
.tab
acum
pPC
VG
luxA
and
Blu
xAan
dlu
xB(V
.ha
rvey
i)lu
xApr
omot
erle
ss,b
uten
hanc
edby
CaM
V35
Spr
omot
erin
fron
tof
luxB
(i.e
.pro
mot
erse
arch
vect
or)
Agr
obac
teri
umtu
mef
acie
nsT
-DN
A-
med
iate
dtr
ansf
erof
lux
into
tran
s-ge
nic
Ara
bido
psis
thal
iana
(RL
D)
for
expr
essi
onin
resp
onse
tovi
ran
dav
irin
fect
ion
byP
seud
omon
assy
ring
ae
Dec
anal
Use
ofph
oton
-cou
ntin
gim
agin
gto
visu
aliz
e(9
3):
�Pr
omot
erse
arch
expr
essi
onof
Lux
Aan
dB
inN
.tab
acum
pLT
u–lu
xFlu
xF(V
.ha
rvey
i)C
rate
rost
igm
apl
anta
gine
umdr
ough
tan
dA
BA
-reg
ulat
edpr
omot
er–l
uxF
Agr
obac
teri
umtu
mef
acie
nsT
-DN
A-
vect
ored
lux
intr
ansg
enic
Nic
otia
nata
bacu
mcv
.SR
1
Dec
anal
Use
ofph
oton
-cou
ntin
gim
agin
gto
visu
aliz
e(9
3):
�St
ress
and
AB
A-i
nduc
edL
uxF
expr
essi
onin
N.t
abac
um
pPC
V70
1–lu
xAB
luxA
B(V
.ha
rvey
i)A
grob
acte
rium
tum
efac
iens
auxi
n-re
gula
ted
man
nopi
nesy
ntha
sebi
-di
rect
iona
lpr
omot
ers
(mas
P1
and
P2)
Tra
nsge
nic
Nic
otia
nata
bacu
mcv
.SR
1D
ecan
alPh
oton
-cou
ntin
gvi
sual
izat
ion
ofau
xin-
indu
ced
activ
atio
nof
mas
prom
oter
sof
luxA
Bin
tran
sgen
icto
bacc
o(9
4)
pWH
1520
–xyl
A–l
uxF,
calle
dpW
H15
20SF
;pL
X70
3-fa
b9lu
xAB
(V.
harv
eyi)
B.m
egat
eriu
mxy
lose
isom
eras
ege
ne(x
ylA
)pr
omot
erlu
x-tr
ansf
orm
edB
acil
lus
thur
ingi
ensi
san
dB
.meg
ater
ium
Dec
anal
Mea
sure
dxy
lose
indu
ctio
nof
xylA
–lux
AB
intr
ansf
orm
edB
acil
lus
thur
ingi
ensi
san
dB
.m
egat
eriu
m(9
5)B
acte
rial
prok
aryo
ticpr
omot
erse
arch
vect
or:
35bp
luxF
–Ori
T–O
riV
–N
PT2–
tran
spos
ase
from
TN
5w
ithka
nr —35
bp
luxF
(V.
harv
eyi)
Ran
dom
prok
aryo
ticpr
omot
erre
gion
sE
.co
liD
ecan
alIm
age-
inte
nsifi
edlo
w-l
ight
sing
le-p
hoto
nvi
deo
imag
ing
(and
X-r
ayau
tora
diog
ra-
phy)
oftr
ansg
enic
orga
nism
sus
ing
bac-
teri
allu
cife
rase
s(9
7):
E.
coli
Vec
tor
cont
aini
ngG
al4
prom
oter
–lux
Flu
xF(V
.ha
rvey
i)G
al4
prom
oter
Sacc
haro
myc
esce
revi
siae
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:ye
ast
Tra
nsge
nic
rhiz
obia
(bac
teri
ods)
cont
aini
ngrh
izob
ium
nitr
ogen
ase
P1pr
omot
er–l
uxF
linka
ge
luxF
(V.
harv
eyi)
Con
stitu
tive
P1
prom
oter
Gly
cine
max
(soy
bean
plan
t)no
dule
sin
fect
edw
ithtr
ansg
enic
Bra
dyrh
izob
ium
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
rhiz
o-bi
umin
soyb
ean
nodu
les
Plan
tex
pres
sion
vect
or:
LB
–p–N
PTII
(neo
myc
inph
osph
otra
nsfe
rase
II)–
pA–p
–lux
F–pA
–RB
pWH
1520
SF–
xylR
–lux
AB
luxF
(V.
harv
eyi)
Aux
in-a
ctiv
ated
man
nopi
nebi
dire
c-tio
nal
1�,2
�(m
as)
prom
oter
s;no
pa-
line
synt
hase
prom
oter
Agr
obac
teri
um-d
eliv
ered
expr
essi
onve
ctor
into
expl
ants
ofN
icot
iana
taba
cum
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
toba
cco
pCV
701–
luxF
luxF
(V.
harv
eyi)
Aux
in-a
ctiv
ated
mas
prom
oter
Agr
obac
teri
um-d
eliv
ered
expr
essi
onve
ctor
into
Lyc
oper
sico
nes
cule
ntum
(tra
nsge
nic
tom
ato
tissu
es)
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
tom
atoe
sPl
ant
prom
oter
sear
chve
ctor
B:
Agr
o-ba
cter
ium
LB
–lux
A–N
OS p
A–a
P CaM
V35S–l
uxB
–NO
S pA
–PN
OS–
HPT
–g7 p
A–A
pr –RB
luxA
B(V
.ha
rvey
i)A
uxin
-act
ivat
edm
as1�
,2�p
rom
oter
sA
grob
acte
rium
-del
iver
edex
pres
sion
vect
orin
toSo
lanu
mtu
bero
sum
(pot
ato)
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
tom
atoe
s
������������ �
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
Imaging of light emission from luciferase expression REVIEW 49
Tab
le2.
Con
tinu
ed
Con
stru
ct/v
ecto
rL
ucif
eras
ege
nes
orcD
NA
sPr
omot
ers/
enha
ncer
sO
rgan
ism
/cel
lsSu
bstr
ate
requ
irem
ent
Imag
ing
appl
icat
ion
and
refe
renc
e
Plan
tpr
omot
erse
arch
vect
orA
:A
gro-
bact
eriu
mT
–DN
Ale
ftbo
rder
,LB–
luxF
–OS p
A–P
NO
S–H
PT–N
OS p
A–
Apr –R
B
luxF
(V.
harv
eyi)
Aux
in-a
ctiv
ated
mas
1�,2
�pro
mot
ers
Agr
obac
teri
um-d
eliv
ered
expr
essi
onve
ctor
into
Sola
num
tube
rosu
m(p
otat
o)
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
tom
atoe
sPl
ant
prom
oter
sear
chve
ctor
B:
Agr
o-ba
cter
ium
LB
–lux
A–N
OS p
A–a
P CaM
V35S–l
uxB
–NO
S pA
–PN
OS–
HPT
–g7 p
A–A
pr –RB
Mas
prom
oter
–lux
AB
(V.h
arve
yi)
fusi
onA
uxin
-act
ivat
edm
as1�
,2�p
rom
oter
;C
aMV
35S
prom
oter
Agr
obac
teri
um–d
eliv
ered
expr
essi
onve
ctor
into
Lyc
oper
sico
nes
cule
ntum
(tra
nsge
nic
tom
ato
frui
t)
Dec
anal
Imag
e-in
tens
ified
low
–lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
tom
ato
frui
tPl
ant
prom
oter
sear
chve
ctor
A:
Agr
o-ba
cter
ium
T–D
NA
left
bord
er,L
B–
luxF
–OS p
A–P
NO
S–H
PT–N
OS p
A–
Apr –R
B
Mas
prom
oter
–lux
F(V
.har
veyi
)fu
sion
Aux
in-a
ctiv
ated
mas
1�,2
�pro
mot
erA
grob
acte
rium
-del
iver
edex
pres
sion
vect
orin
toN
icot
iana
taba
cum
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
toba
cco
stem
sPl
ant
prom
oter
sear
chve
ctor
A:
Agr
o-ba
cter
ium
T–D
NA
left
bord
er,L
B–
luxF
–OS p
A–P
NO
S–H
PT–N
OS p
A–
Apr –R
B
Mas
prom
oter
–lux
F(V
.har
veyi
)fu
sion
Aux
in-a
ctiv
ated
mas
1�,2
�pro
mot
erA
grob
acte
rium
-del
iver
edex
pres
sion
vect
orin
toD
atur
ast
ram
oniu
mD
ecan
alIm
age-
inte
nsifi
edlo
w-l
ight
sing
le-p
hoto
nvi
deo
imag
ing
(and
X-r
ayau
tora
diog
ra-
phy)
oftr
ansg
enic
orga
nism
sus
ing
bac-
teri
allu
cife
rase
s(9
7):
tran
sgen
icD
atur
aB
acte
rial
prok
aryo
ticpr
omot
erse
arch
vect
or:
35bp
luxF
–Ori
T–O
riV
–N
PT2–
tran
spos
ase
from
Tn5
with
kanr —
35bp
luxF
(V.
harv
eyi)
Ran
dom
prok
aryo
ticpr
omot
erre
gion
sP
seud
omon
asso
lana
caer
umvi
aco
n-ju
gatio
nw
ithan
E.
coli
bear
ing
Tn5
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
Pse
udo-
mon
as—
visu
aliz
ing
Plan
tpr
omot
erse
arch
vect
orB
:A
gro-
bact
eriu
mL
B–l
uxA
–NO
S pA
–aP C
aMV
35S–l
uxB
–NO
S pA
–PN
OS–
HPT
–g7 p
A–A
pr –RB
Het
erod
imer
iclu
xAB
(V.
harv
eyi)
Aux
in-a
ctiv
ated
mas
1�,2
�pro
mot
eror
phen
ylal
anin
eam
mon
ialy
ase
(PA
L)
prom
oter
Tra
nsge
nic
Sola
num
tube
rosu
m(p
otat
o)in
fect
edw
ithE
rwin
iahe
rbic
ola
(avi
rule
nt),
E.
caro
tovo
ra(v
irul
ent)
,Pse
udom
onas
syri
ngae
stra
into
mat
o(a
viru
lent
)
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
Pse
udo-
mon
as—
mon
itori
ngth
evi
rule
nce
ofpa
thog
enst
rain
spP
CV
701–
luxA
Blu
xAB
(V.
harv
eyi)
Aux
in-a
ctiv
ated
mas
1�,2
�pro
mot
ers
Tra
nsge
nic
Sola
num
tube
rosu
m(p
otat
o)in
fect
edby
Pse
udom
onas
syri
ngae
,Erw
inia
caro
tovo
raca
ro-
tovo
ra,E
rwin
iaca
roto
vora
atro
sep-
tica
,E
rwin
iahe
rbic
ola
Dec
anal
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
(97)
:tr
ansg
enic
pota
to
pWH
1520
SF–x
ylR
–xyl
A–l
uxA
Blu
xAB
(V.
harv
eyi)
–xyl
Apr
omot
erfu
sion
xylA
prom
oter
Bac
illu
sth
urin
gien
sis
and
Bac
illu
sm
egat
eriu
mD
ecan
alIm
age-
inte
nsifi
edlo
w-l
ight
sing
le-p
hoto
nvi
deo
imag
ing
(and
X-r
ayau
tora
diog
ra-
phy)
oftr
ansg
enic
orga
nism
sus
ing
bac-
teri
allu
cife
rase
s(9
7):
Bac
illu
sA
cNPV
–luc
luc
AcN
PVpo
lyhe
drin
late
prom
oter
Aut
ogra
pha
cali
forn
ica
nucl
ear
poly
-he
dros
isvi
rus-
vect
ored
Luc
inT
.ni
368
cells
and
Tri
chop
lusi
ani
3rd
inst
arla
rvae
Fire
flylu
cife
rin
Imag
e-in
tens
ified
low
-lig
htsi
ngle
-pho
ton
vide
oim
agin
g(a
ndX
-ray
auto
radi
ogra
-ph
y)of
tran
sgen
icor
gani
sms
usin
gba
c-te
rial
luci
fera
ses
and
firefl
ylu
cife
rase
(97)
:T
.ni
368
cells
and
T.n
i3r
din
star
larv
aelu
xAan
dB
(V.
harv
eyi)
,Fab
9,F
abcb
c9–
E.
coli
Dec
anal
Imag
ing
ofov
erex
pres
sion
ofG
roE
Lan
dG
roE
S-m
edia
ted
fold
ing
ofFa
b9–b
acte
rial
luci
fera
sefu
sion
sat
diff
eren
tte
mpe
ratu
res
(97)
:E
.co
lipC
EP4
–luc
luc
CM
Vpr
omot
erB
rach
ydan
iore
rio
(zeb
rafis
h)L
ucif
erin
(fire
fly;
0.1
mm
ol/L
)T
rans
geni
cze
brafi
shw
ithL
ucvi
sual
ized
bylo
w-l
ight
vide
o-im
age
anal
ysis
(195
)pP
VC
701–
ruc
ruc
(Ren
illa
reni
form
islu
cife
rase
)pC
aMV
35S
RN
AA
lfal
fa(M
edic
ago
sati
va),
tom
ato,
pota
to,t
obac
coC
oele
nter
azin
eIm
agin
gof
Ruc
intis
sues
oftr
ansg
enic
plan
ts(6
0)pM
W54
;pM
V53
;pM
V16
;pO
GS2
13lu
cH
IV-1
,HIV
-1LT
Ren
hanc
er–p
rom
o-er
elem
ents
and
CM
Vpr
omot
erH
eLa
cells
Luc
ifer
in(fi
refly
)V
isua
lizat
ion
ofH
IV-
and
hCM
V-p
rom
oted
expr
essi
onof
Luc
insi
ngle
mam
mal
ian
(HeL
a)ce
lls(1
39)
pCol
.luc;
pCM
V.lu
c;pC
MV
.Aqm
;pc
DN
AA
Ineo
.CL
100
luc;
aequ
orin
Col
lage
nase
and
CM
Vpr
omot
ers
CH
O.T
cells
Coe
lent
eraz
ine
and
beet
lelu
cife
rin
(for
Aeq
and
Pluc
resp
ectiv
ely)
Hig
h-in
tens
ityre
al-t
ime
phot
on-c
ount
ing
imag
ing
ofin
sulin
-ind
uced
MA
Pki
nase
sign
alin
gin
sing
lece
lls(1
37)
������������ �
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
50 REVIEW L. F. Greer and A. A. Szalay
Tab
le2.
Con
tinu
ed
Con
stru
ct/v
ecto
rL
ucif
eras
ege
nes
orcD
NA
sPr
omot
ers/
enha
ncer
sO
rgan
ism
/cel
lsSu
bstr
ate
requ
irem
ent
Imag
ing
appl
icat
ion
and
refe
renc
e
pGL
101
luxF
(lux
AB
fusi
on)
Ara
bido
psis
PA
L1
(phe
nyla
lani
neam
mon
ia-l
yase
)pr
omot
erA
rabi
dops
isth
alia
naD
ecan
alSu
cces
sful
phot
on-c
ount
ing
imag
ing
oflo
caliz
edac
tivat
ion
ofPA
L1
(187
)pC
EP4
ruc–
gfp
fusi
on,a
ndin
divi
dual
lyC
MV
and
hum
an�-
actin
prom
oter
sL
M-T
K�
cells
;m
urin
eem
bryo
nic
stem
(ES)
cells
grow
th-s
uppo
rted
byST
Ofe
eder
cells
Coe
lent
eraz
ine
Vis
ualiz
atio
nof
Ruc
–GFP
expr
essi
onin
ES
cells
and
embr
yos
(69)
pCE
P4–R
uc;
pCE
P4–R
uc/G
FP;
pCE
P4–G
FP/R
uc;
pGE
M–5
z(�)
–Ruc
/G
FP;
pGE
M-5
z(�)
–GFP
/Ruc
ruc;
‘hum
aniz
ed’
gfp
(Aeq
uore
a)C
MV
,�-a
ctin
,(f
orpC
EP4
vect
or),
and
T7
(pG
EM
-5zf
�ve
ctor
)pr
omot
ers
LM
-TK�
(mur
ine
fibro
blas
tce
lllin
ew
ithth
ymid
ine
kina
sem
issi
ngbe
caus
eof
mut
atio
n)
Coe
lent
eraz
ine
Imag
ing
Ruc
–mod
ified
GFP
fusi
onin
mur
ine
cells
(70)
Mito
chon
dria
llyta
rget
edae
quor
inve
c-to
rsae
q(A
equo
rea)
–C
HO
.Tce
llsC
oele
nter
azin
eIm
aged
intr
amito
chon
dria
lC
a2�
ince
llsus
ing
reco
mbi
nant
aequ
orin
with
aC
CD
cam
era
(138
)R
ecom
bina
ntba
culo
viru
sco
nstr
ucts
Bm
NPV
luc
(Bom
byx
mor
inu
clea
rpo
lyhe
dros
isvi
rus)
and
AcN
PVlu
c[2
]
luc
[2]
Inre
vers
eor
ient
atio
nto
the
Aut
o-gr
apha
cali
forn
ica
nucl
ear
poly
-he
dros
isvi
rus
(AcN
PV)
poly
hedr
invi
ral
prom
oter
,pos
sibl
yun
der
OR
F629
prom
oter
Bom
byx
mor
iN
-4an
dSf
9ce
lls,
Tri
chop
lusi
ani
368
cells
Luc
ifer
in(fi
refly
)Im
agin
gof
bacu
lovi
rus-
vect
ored
Luc
inin
sect
cells
(218
)
pRL
uc6
and
pRL
uc6.
1;pM
CT
–Ruc
ruc
AdV
maj
orla
tepr
omot
er;
hCM
Vin
term
edia
te–e
arly
enha
ncer
/pr
omot
er
CO
S-7
cells
;C
5ce
llsC
oele
nter
azin
eIm
agin
gof
Ruc
expr
essi
onin
sim
ian
and
mur
ine
cells
(61)
Rec
ombi
nant
herp
es/
pseu
dora
bies
viru
sPr
VA
916
luc
Gly
copr
otei
ngG
earl
ypr
omot
erA
fric
angr
een
mon
key
kidn
ey(V
ER
O)
cells
Luc
ifer
inV
isua
lizin
gof
PrV
infe
ctio
nin
cultu
revi
aPl
ucus
ing
aph
oton
-co
untin
gca
mer
a(2
17)
CH
S::lu
cco
nstr
uct
luc
Frag
men
tof
chal
cone
synt
hase
prom
o-te
r;�
tran
slat
iona
len
hanc
erof
the
Tob
acco
Mos
aic
Vir
us(T
MV
)
Ath
alia
nase
edlin
gs(C
olum
bia
g/1)
and
inan
A.
thal
iana
C24
cell
line
Luc
ifer
inC
ompa
riso
nsbe
twee
nai
r-co
oled
CC
Dan
din
tens
ified
CC
Dca
mer
as(1
78)
LT
R–l
ucin
pGL
3lu
cSV
40pr
omot
eran
den
hanc
erin
LT
Rs
Neo
nata
lra
ts(R
attu
s);
mic
e(M
us);
hum
anT
cells
Luc
ifer
in(fi
refly
)In
duct
ion
ofSV
40pr
omot
er–l
ucif
eras
eex
pres
sion
inne
onat
alra
ts,i
nm
ice/
and
inhu
man
T-c
ells
(157
)pL
PKL
ucF
F,t
runc
ated
p�L
4–L
PKL
ucF
F
ruc
L-p
yruv
ate
kina
seno
rmal
ized
byco
n-st
itutiv
eC
MV
prom
oter
,req
uiri
ngup
stre
amst
imul
ator
yfa
ctor
2(U
SF2)
Hum
anis
let�-
cells
,der
ived
INS-
1ce
llsB
eetle
luci
feri
n,eo
elen
tera
zine
Sing
le-c
ell
CC
Dim
agin
gof
Ruc
-mar
ked
nece
ssar
yup
stre
amst
imul
ator
yfa
ctor
activ
ity(1
36)
pCK
218
luxA
B(V
ibri
ofis
cher
i)Pr
omot
erle
ssin
V.
fisch
eri;
unid
enti-
fied
stro
ngco
nstit
utiv
epr
omot
erin
Pse
udom
onas
puti
da
Vib
rio
fisch
eri
MJ-
1;lu
x-m
arke
dP
seud
omon
aspu
tida
Dec
anal
Com
pari
son
ofsi
ngle
-ba
cter
ium
low
-lig
htim
agin
gus
ing
acr
yoge
nica
lly-c
oole
dC
CD
cam
era
and
aph
oton
-cou
ntin
gca
mer
a(1
41)
pTK
Elu
c;pT
K6W
Elu
c;pS
VE
luc;
pMiw
luc;
pMiw
Elu
clu
c6W
enha
ncer
,TK
;SV
40;
RSV
(Rou
sSa
rcom
aV
irus
)L
TR
,�-a
ctin
prom
oter
s
Bov
ine
embr
yos
Luc
ifer
in(fi
refly
)D
etec
tion
oflig
htem
issi
onfr
omL
ucin
tran
sgen
icbo
vine
embr
yos
(200
)
–ru
c(R
enil
lare
nifo
rmis
)Pr
omot
erle
ssR
ucex
pres
sion
–pro
mot
erse
arch
vect
orT
rans
geni
cA
rabi
dops
is,T
abac
umor
othe
rpl
ant
calli
,ro
ots,
leaf
,st
em,
flow
ertis
sue;
pota
totu
bers
;se
di-
men
ted
tran
sfor
med
plan
tpr
oto-
plas
ts(l
ux,r
uc);
tran
sgen
ic
Dec
anal
;lu
cife
rin
2-be
nzyl
coel
ente
razi
neIm
agin
gof
plan
tpr
omot
erex
pres
sion
usin
gL
uxan
dR
ucre
port
ers
inpl
ant
cells
(182
)
pSP–
Luc
�ph
PRL
luc
hPR
L(h
uman
prol
acti
n)pr
omot
erR
atpi
tuita
rytu
mou
rG
H3
cells
Luc
ifer
in(fi
refly
)C
CD
phot
on-c
ount
ing
imag
ing
ofPR
Lpr
omot
erac
tivat
ion
ofL
ucin
indi
vidu
alce
lls(1
21)
RD
29A
–Luc
cons
truc
tlu
cR
D29
A;
CO
R47
;C
OR
15A
;K
IN1;
AD
H;
RA
B18
;R
D22
;R
D29
B;
LUC
;ac
tin—
all
cold
sens
itive
gene
prom
oter
s
Ara
bido
psis
thal
iana
Luc
ifer
in(fi
refly
)V
isua
lized
mut
ant
seed
lings
with
mut
ant
cold
-res
pons
ege
neH
OS-
1(1
81)
Synt
hetic
RB
CSB
gene
luc
(pro
mot
erle
ssin
cons
truc
t)R
BC
SBpr
omot
erju
xtap
osed
upst
ream
oflu
cby
cros
s-ov
erA
rabi
dops
isth
alia
naL
ucif
erin
(fire
fly)
Vis
ualiz
edcr
oss-
over
seed
lings
(184
)
p260
Ins.
Luc
FF;
pF71
1fo.
Luc
FF;
pCM
V–R
enlu
c;ru
cSR
Ean
dC
RE
ofth
ehu
man
insu
linpr
omot
er,H
erpe
ssi
mpl
exm
inim
alTK
,c-f
osan
dC
MV
prom
oter
s
MIN
6�-
cells
;C
HO
cells
;an
teri
orpi
tuita
ry-d
eriv
edA
tT20
Luc
ifer
in(fi
refly
);co
elen
tera
zine
Rea
l-tim
ein
tens
ified
CC
Dca
mer
aim
agin
gof
cons
titut
ive
gluc
ose
enha
ncem
ent
ofin
sulin
prom
oter
-act
ivat
edlu
cife
rase
activ
ity(1
23)
pcL
uc(n
on-t
arge
ted,
cyto
solic
luci
fer-
ase)
;pm
Luc
(pla
sma
mem
bran
eta
rget
edlu
cife
rase
)
luc
CM
Van
dSV
40in
term
edia
te–e
arly
prom
oter
sPr
imar
yra
tis
let�-
cells
;de
rive
dM
IN6
cells
Luc
ifer
in(fi
refly
)L
ucif
eras
eph
oton
-cou
ntin
gim
agin
gof
sub-
cellu
lar
com
part
men
tali
zatio
nof
AT
P(1
24)
������������ �
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
Imaging of light emission from luciferase expression REVIEW 51
Tab
le2.
Con
tinu
ed
Con
stru
ct/v
ecto
rL
ucif
eras
ege
nes
orcD
NA
sPr
omot
ers/
enha
ncer
sO
rgan
ism
/cel
lsSu
bstr
ate
requ
irem
ent
Imag
ing
appl
icat
ion
and
refe
renc
e
RD
29A
–Luc
luc
RD
29A
prom
oter
Tra
nsge
nic
Ara
bido
psis
(eco
type
C24
)tr
ansf
orm
edby
Agr
obac
teri
umtu
mef
acie
ns
Luc
ifer
in(fi
refly
)R
eal-
time
ther
moe
lect
rica
llyco
oled
CC
Dca
mer
avi
sual
izat
ion
ofL
uc-m
arke
dst
ress
resp
onse
inA
rabi
dops
is(1
85,1
86)
pGL
3M
odifi
edlu
cSV
40pr
omot
erH
eLa-
luc
cells
inyo
ung
CB
17SC
IDm
ice
Luc
ifer
in(fi
refly
)V
isua
lizat
ion
ofH
eLa–
Luc
tum
ours
resu
lt-in
gfr
omin
trap
erito
neal
inje
ctio
n(1
60)
pGL
3—so
urce
ofL
uclu
cM
urin
ehe
me
oxyg
enas
e1
(HO
-1)
prom
oter
Tra
nsge
nic
mic
ew
ith(H
O–l
uc;
mur
ine
hem
eox
ygen
ase
1–Pl
uc)
Luc
ifer
ine
(fire
fly)
Inte
nsifi
edC
CD
cam
era
mon
itori
ngof
Pluc
expr
essi
onas
saye
dle
vels
oftis
sue
oxyg
enat
ion
intr
ansg
enic
mic
e(2
01)
pMK
4lu
xAB
CD
Elu
xAB
CD
E(i
nter
sper
sed
with
Gra
m-
posi
tive
ribo
som
ebi
ndin
gsi
tes)
Ran
dom
lyin
tegr
ated
into
S.au
reus
geno
me—
diff
erin
gex
pres
sion
leve
ls—
prom
oter
s
Stap
hylo
cocc
usau
reus
inm
ice
Non
ene
cess
ary—
full
lux
oper
on(s
cree
ning
done
with
n-de
cyl
alde
hyde
)
Succ
essf
ulim
agin
gof
Gra
m-p
ositi
veba
cter
ial
Lux
inm
ice
(202
)
pHA
L11
9;m
ini-
Tn1
0lux
AB
cam
/Pt
ac–A
TS
luxA
B(V
.ha
rvey
i)Pr
omot
erle
ssin
plas
mid
,but
tran
sduc
eddo
wns
trea
mof
E.c
oli
prom
oter
s
Esc
heri
chia
coli
O15
7:H
7D
ecan
alV
isua
lizin
gE
.co
lico
loni
estr
ansd
uced
byT
nan
dL
uxex
pres
sion
AB
byim
age
quan
tifier
(101
)pL
PK–L
ucF
F;
p�L
4–L
PK–L
ucF
F;
pIN
S–lu
c FF;
pGL
3ba
sic;
pRL
-CM
Vlu
c;hu
man
ized
luc;
ruc
Rat
L–P
K(l
iver
-typ
epy
ruva
teki
nase
);hu
man
insu
lin;
Her
pes
sim
plex
min
imal
TK
and
CM
Vpr
omot
ers
Panc
reat
ic(M
IN6)
�-is
let
cells
Bee
tlelu
cife
rin,
coel
ente
razi
neIm
agin
gof
AM
P-ac
tivat
edPK
insi
ngle
cells
usin
glu
cife
rase
expr
essi
on(1
26)
pLPK
-Luc
FF;
p(�1
50)
LPK
-Luc
FF;
pIN
S-L
ucF
F;
pRL
-CM
V;
p�G
K4-
Luc
;A
dCM
VcL
uc;
pPPI
-L
ucF
F
luc;
hum
aniz
edlu
c;ru
cR
atL
-PK
,hum
anin
sulin
,Her
pes
sim
plex
min
imal
TK,C
MV
imm
edia
te-e
arly
,rat
�-ce
llgl
uco-
kina
se,a
ndhu
man
PP
Ipr
omot
ers
Panc
reat
ic(M
IN6)
�-is
let
cells
Bee
tlelu
cife
rin,
coel
ente
razi
neSi
ngle
-cel
lim
agin
gof
gluc
ose-
activ
ated
insu
linse
cret
ion
and
activ
atio
nof
phos
-ph
atid
ylin
osito
l3-
kina
seus
ing
luci
fera
seex
pres
sion
(127
)pN
D2-
Ruc
/GFP
;pN
D2-
Sruc
/GFP
ruc/
gfp;
secr
eted
ruc/
gfp
(Sru
c/gf
p)C
MV
prom
oter
CO
S-7;
CH
Oce
llsC
oele
nter
azin
eIm
agin
gan
dqu
antif
ying
ofR
uc–G
FPfu
sion
prot
ein
secr
etio
n(7
3)pG
L3
luc
–9L
Luc
rat
glio
sarc
oma
cells
invi
voin
Fisc
her
344
rats
Luc
ifer
in(fi
refly
)A
sses
smen
tof
chem
othe
rape
utic
prog
ress
intr
eatin
gL
uc-b
eari
ngtu
mor
cells
thro
ugh
imag
ing
inra
ts(2
08)
Tn4
001
luxA
BC
DE
Km
r ;pA
UL
-AT
n400
1lu
xAB
CD
EK
mr
(Gra
m�)
luxA
BC
DE
Km
rop
eron
(Pho
to-
rhab
dus
lum
ines
cens
;G
ram�)
Prom
oter
less
-a
prom
oter
sear
chve
ctor
:R
ando
mtr
ansp
oson
inse
rtio
nin
toth
eS.
pneu
mon
iae
geno
me
be-
hind
stro
nger
orw
eake
rpr
omot
ers
Lux
-tra
nsfo
rmed
Gra
m�
Stre
pto-
cocc
uspn
eum
onia
ein
fect
ion
inB
AL
B/c
mic
e
NA
—lu
xop
eron
prod
uces
itsow
nsu
bstr
ate
Tra
nsfo
rmed
lum
ines
cent
,kan
amyc
inre
sis-
tant
bact
eria
wer
eno
n-in
vasi
vely
visu
al-
ized
invi
voin
mic
e;C
CD
imag
ing
oflo
nger
-ter
mpn
eum
ococ
cal
lung
infe
ctio
nsin
mic
eus
ing
bact
eria
tran
sfor
med
with
the
Gra
m-p
ositi
velu
xtr
ansp
oson
(214
)A
dCM
VL
uc;
Ad5
Luc
RG
Dlu
cC
MV
prom
oter
A54
9ce
llsC
ell
mem
bran
e-pe
rmea
ble
acet
oxy
met
hyl
este
rde
riva
tive
ofD
-luc
ifer
in
Imag
ing
ofPl
ucex
pres
sion
toas
say
diff
eren
ces
insi
gnal
tran
sduc
tion
effic
ien-
cies
oftw
oA
dve
ctor
sw
ithdi
ffer
ent
cell
bind
ing
affin
ities
(219
)A
AV
–EF1
�–lu
cife
rase
(rA
AV
);pS
SV9–
E1�
–luc
ifer
ase;
pXX
2;pX
X6
luc
EF
1�pr
omot
erM
ice
(CD
-1)
Luc
ifer
in(fi
refly
)Im
agin
gof
long
-ter
min
trap
erit
onea
lL
ucex
pres
sion
vect
ored
byrA
AV
inm
ice
(165
)pE
T–G
2R;
pET
–RG
2;pC
–IG
F–II
–G
FP;
pC–I
GFB
P6–R
uc;
pC–
IGFB
P6–R
uc;
pC–I
NS–
GFP
ruc–
gfp
(Aeq
uore
a)fu
sion
T7
and
CM
Vpr
omot
ers
Sim
ian
CO
S-7
cells
Coe
lent
eraz
ine
Inte
nsifi
edC
CD
cam
era
mag
ing
oflu
min
esce
nce
reso
nanc
een
ergy
tran
sfer
(LR
ET
)fr
omR
ucto
Aeq
GFP
(72)
AdC
MV
cLuc
;pc
Luc
;A
dPPI
cLuc
;pm
tAE
Q;
AdC
MV
mL
uc;
pAdT
rack
CM
V;
pAD
CM
Vm
Aq
Aeq
uori
n(a
eq);
luc
(hum
aniz
ed)
CM
Van
dhu
man
prep
roin
sulin
(PP
I)pr
omot
ers
MIN
6�-
isle
tce
llsL
ucif
erin
(fire
fly)
Imag
ing
ofch
ange
sin
Ca2
�an
dA
TPc
inin
divi
dual
cells
via
luci
fera
sean
dae
quor
inA
dCM
VcL
uc(1
30)
rVV
–RG
[rV
V–P
E/L
–ruc
–gfp
]R
enil
lalu
cife
rase
(ruc
)V
acci
nia
stro
ngsy
nthe
ticea
rly–
late
(PE
/L)
prom
oter
CV
-1A
fric
angr
een
mon
key
kidn
eyce
lls;
athy
mic
nu/n
um
ice
Coe
lent
eraz
ine
Low
-lig
htim
agin
gof
reco
mbi
nant
Vac
cini
avi
ral
infe
ctio
nin
cell
cultu
rean
dim
mun
e-co
mpr
omis
edm
ice
(222
)pA
M40
1ASG
XpS
B20
35[–
xylA
–gfp
–lux
AB
CD
E]
luxC
DA
BE
(Pho
torh
abdu
slu
mi-
nesc
ens)
fuse
dto
gfp
(Aeq
uore
a)lu
xCD
AB
E(P
hoto
rhab
dus
lum
i-ne
scen
s);
Aeq
uore
agf
p
SOD
(sup
erox
ide
dism
utas
e)pr
omot
erxy
lApr
omot
erE
.co
lian
dot
her
Gra
m-n
egat
ive
bact
eria
;vi
sual
ized
innu
/nu
mic
eB
ovin
em
amm
ary
epith
elia
lM
AC
-Tce
lls
Non
eN
one
Imag
ing
ofba
cter
ial
infe
ctio
nin
mic
ean
dra
ts(2
21)
Vis
ualiz
ing
the
expr
essi
onan
dte
mpo
ral
indu
ctio
nof
the
quor
um-s
ensi
ngac
ces-
sory
gene
regu
lato
r(A
gr)
inS.
aure
usin
MA
C-T
cells
(133
)A
d–C
MV
–Luc
luc
CM
Vpr
omot
erM
uscl
esof
imm
unoc
ompe
tent
Swis
sW
ebst
erm
ice
Vis
ualiz
ing
the
loca
tion,
mag
nitu
dean
dpe
rsis
tenc
eof
Luc
expr
essi
onin
mic
eby
CC
CD
imag
ing
(203
)A
dSV
40/L
uc;
AdH
IV/L
uc;
rLN
C/L
uc;
pLN
C/L
uclu
cC
MV
,C/E
BP�
(PL
AP),
SV40
,CM
V,
BG
LAP
(ost
eoca
lcin
),H
IV–L
TR
,SV
40an
dC
MV
,SV
40an
dH
19pr
omot
ers
Tra
nsfe
cted
cell
lines
:H
epG
2/L
uc(h
uman
hepa
toce
llula
rca
rcin
oma)
;PC
3.38
/Luc
(clo
neof
hum
anhu
man
pros
tate
aden
ocar
cino
ma)
;T
50/L
uc
Bee
tlelu
cife
rin
ICC
Dan
dC
CC
Dvi
sual
izat
ion
ofL
ucex
pres
sion
inliv
ing
anim
als
unde
rva
riou
sco
nditi
ons
and
para
met
ers
toop
timiz
elu
cife
rase
invi
voim
agin
g(2
09)
������������ �
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
52 REVIEW L. F. Greer and A. A. Szalay
photoproteins as simultaneous reporters will probablybecome more common in the future.
�� ���
The papers cited above show that luciferases have beenvery useful as reporter genes in living cells and inbioluminescent immunoassays (63). In fact there wereabout 30 photoprotein fusions and conjugates reportedbetween 1988 and 2000 (63–65). The first such fusion wasthat of the Vibrio harveyi luxA and luxB into the luxAB(luxF) fusion, which was expressed as a monomer in E. coli,Bacillus, yeast and plant systems (30, 66). A luxAB fusionhas also been made from the luciferase of Photorhabdusluminescens (67). A gfp–luxAB fusion construct expressedin E. coli DH5� and Pseudomonas fluorescens SBW25 wasused to assay bacterial numbers and nutrient-based bacterialmetabolic activity in soil samples (68). Assaying the luxABexpression required luminometry (For more information,see section on Bacterial luciferase imaging in environ-mental health assays, below).
An ruc–gfp fusion construct was first engineered andexpressed in murine LM–TK� fibroblast cells, inembryonic stem (ES) cells and in early stage embryosby Wang et al. (1996) (69). A ruc-modified gfp fusionwas found to be functionally expressed in murine LM–TK� cells, whereas a reverse modified gfp–ruc fusionshowed no GFP expression, probably because ofmisfolding (70). In 1999, Wang et al. suggested thatchemiluminescent energy transfer Ruc to GFP could beused to image protein–protein interactions (71).
Using an ruc–gfp fusion construct, Wang et al. (2001)imaged the luminescence resonance energy transfer(LRET) phenomenon from Renilla luciferase (Ruc)emission to a humanized Aequorea GFP to documentprotein–protein interaction in eukaryotic cells (72).cDNAs of ruc and insulin-like growth factor bindingprotein 6 (Ruc–IGFBP-6) were expressed, along withfused cDNAs for gfp and insulin-like growth factor II(gfp–igf-II). The interaction of the recombinant IGF-IIand IGFBP-6 resulted in LRET (luminescence resonanceenergy transfer) from Ruc to GFP. In 2000, Liu et al.visualized and quantified protein secretion using an Ruc–GFP fusion in the COS-7 and Chinese hamster ovary(CHO) cell lines (73). In simian COS-7 cells, intensifiedCCD camera imaging has been used to detect LRET fromRuc to aequorin GFP in an Ruc–GFP fusion protein (72).
An important recent development is the use of P.luminescens luxCDABE gene fusions in a collection of8066 individual E. coli transformants to assay genome-wideexpression profiles in response to environmental stress (74).
�#21���.! �'��� � !(&. #�#�/
In 1989, Wick (75) reviewed the growing usage of single
photon-counting visualization being used to assay lumi-nescence in microtitre plates, to image metabolitedistribution in tumour tissues, to visualize single cellgene expression, and even to visualize the faint chemi-luminescence resulting from oxidative metabolism inphagocytes.
In a series of papers from 1992 to 2000, Stanleyreviewed the commercially available luminometers,radiometers, low-light imaging CCD cameras, immu-noassays, ATP rapid microbiology, hygiene monitoring,molecular probes, labels, nucleic acid hybridizations andreporter genes available for bio-imaging applications, aswell as units and standards of bioluminescence (76–86).
Hill and Stewart (1994) reviewed the applied devel-opments of bacterial luciferases as real-time, non-invasive reporters using low-light and photon-countingvideo cameras (87). They noted their sensitivity and real-time, non-invasive nature and their amenability toimaging by photon-counting and low-light video cam-eras. Contag et al. published two reviews on the variousmolecular imaging technologies for the detection andtracking of molecules and cells in vivo (88, 89). They alsodescribed briefly the work done in imaging tumours usingluciferase expression in vivo.
It is to the imaging experiments that we now turn (seeTable 2).
�--��&�!�# � #� �%&��("��( �'��� �
We attempt to summarize the imaging of luciferaseexpression in individual cells and cell cultures, inindividual bacteria, yeasts, algae, insect cells, plant cells,or mammalian cells. We also focus on some of theapplications and types of questions that can be answeredby such imaging. These imaging experiments include theimaging of luciferases in transformed cells and cellsystems in real time.
Next, we consider imaging of luciferase expression inmulticellular organisms in vivo. This includes theexpression and visualization of luciferase in permanentlytransformed or transgenic organisms.
Finally, we present representative studies usingluciferase expression imaging to investigate host–patho-gen interactions in whole plant and animal models.
�'��� � #� �%&��("��( (3-"(���# � !"� ��#"'($ �� ��( &(��� � $ &(��&%�!%"(�
������� �� ���4������� 56�������7 ������� � ���� ��� �� ������
Imaging of bacterial luciferase in bacteria and cell culturehas proved to be a fruitful venture. Some representative
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
Imaging of light emission from luciferase expression REVIEW 53
papers are overviewed below. The monitoring ofbioluminescent bacterial pathogens and symbionts inhosts is discussed in the section on symbiosis and host–pathogen interactions.
In a landmark paper, Engebrecht and his co-workersshowed, in 1985, that the bacterial luciferase operonluxCDABE (i.e. luxICDABE under the control of luxRand luxI) from Vibrio fischeri could be vectored by atransposon and expressed in E. coli without the need toadd the lux substrate, decanal, because of the presence ofthe full operon (26). Ulitzur and Khun (1987) alsodiscussed the use of introduced luciferase genes inbacteria as an assay for the presence of particular bacteriaand their susceptibility to a given antibiotic (90). In 1988,Schauer et al. visualized Vibrio harveyi luxA and luxBexpression in Streptomyces coelicolor (91). Olsson et al.(1988) have shown that fusion gene products can beadded to the luxA of Vibrio harveyi as long as the N-terminal hydrophobic sequences of the �-subunit arepreserved intact, in order to retain enzymatic activity(29).
Olsson et al. (1989) constructed luxAB and luxBAfusions of the V. harveyi luciferase genes and expressedthem in E. coli (Fig. 1) and in calli of Nicotiana tabacum,indicating their possible application as reporter genes ineukaryotic cells (30). The authors showed that luxAB hashigher expression levels than luxBA. This was animportant first. Escher et al. (1989) described a fusionof the luxAB genes of V. harveyi and showed it to becapable of functioning as a monomer in E. coli (66).Using video-imaging and spectroscopy, they found thatluxAB has an emission spectrum comparable to the wild-type luxA and luxB, but is more sensitive to elevatedtemperature. In 1991, Langridge et al. provided anoverview of the bacterial luciferase gene expression
system and its applications, using low-light imaging inother bacteria and in eukaryotic cells, namely plant cells(92).
In a series of studies (93–97) it was shown that Vibrioharveyi luxA, luxB and a luxAB fusion could besuccessfully expressed under various bacterial, mamma-lian and viral promoters and visualized by photon-counting imaging in Gram-positive Bacillus thuringien-sis and B. megaterium (cf. Fig. 2), Arabidopsis thaliana,Nicotiana tabacum, Trichoplusia ni (cabbage looper)(386) cells and Manduca sextans (tobacco hornworm;Fig. 3a, b; see the section a Imaging of host–pathogeninteractions, below) (95). These lux constructs werevectored into transgenic plants by Agrobacteriumtumefaciens T-DNA. Through T-DNA integration, pro-moterless constructs bearing luxA and luxB wererandomly inserted into transgenic Nicotiana tabacum.Lux activity was visualized at different developmentalstages in different organs (Fig. 4) (96). Bacterialluciferase expression was also successfully imaged intomato leaves and fruit (Fig. 5a, b) (97). Firefly luciferase(Pluc) was also expressed in some of these experiments.
When promoterless V. harveyi luciferase (luxAB) wasintroduced downstream of the promoter for the cyano-bacterium Synechococcus psbAI gene (a photosystem IIprotein), its varying expression under constant bio-luminescent imaging revealed that prokaryotes alsohave circadian rhythms (98). Furthermore, when luxABwas inserted randomly by conjugation and subsequenthom-ologous recombination into the Synechococcusgenome and transformed clones were monitored by thethen newly developed cooled-CCD camera system, itwas found that luciferase expression in these cyano-bacteria exhibited not only circadian rhythmicity but a
Figure 1. Visualization of individual Escherichia colicolonies transformed by Vibrio harveyi lux genes (cf. 97).
Figure 2. Visualization of lux-transformed Pseudomonaspatches grown on agar medium using the Argus-100 low-lightimaging system. (cf. 97).
Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74
54 REVIEW L. F. Greer and A. A. Szalay
wide variety of amplitude and waveform cyclicity (99).Liu et al. also discovered that random insertion ofpromoterless luciferases by homologous recombinationis an extremely sensitive assay for differential geneexpression levels. Thus, bacterial luciferase imaging wasuseful in documenting a complex gene expressionphenomenon such as cyanobacterial circadian rhythms(100).
In 2000, Kunert et al. transfected Synechocystis withgfp and luxAB (64). Under different media conditions,luminescence imaging revealed that GFP expressioninitiated and dissipated at a slower rate, while LuxABexpression had a much more rapid response reactiontime. The advantage of GFP in Synechocystis is that it hasno substrate requirement. The advantage of LuxAB is itsrapid expression response time. So, in combination withanother reporter gene, such as GFP, LuxAB can be usedas a sensitive measure of gene expression in bacteria.
Waddell and Poppe (2000) developed a mini-transpo-son bearing Vibrio harveyi luciferase (luxAB) in order toproduce a luciferase-transfecting bacteriophage to detectE. coli strain 0157:H7 (101). The E. coli colonies ofinterest could be visualized by image quantifier about 1 hpost-transfection. The ability to use a vectored luxAB todetect the presence of bacteria has led to importantenvironmental and food safety applications, of which wereview a few.
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One of the earliest uses of luciferase imaging to detectmicrobial contamination involved eukaryotic firefly(Photinus) luciferase (Luc), not bacterial luciferase
Figure 3. Visualization of the progression of an infection ofBacillus thuringiensis, labelled with the xylA promoter–luxABfusion gene construct, in tobacco hornworm (Manduca sextans)larvae (A) through feeding or (B) after injection into thehaemolymph (95). Figure 4. Visualization of randomly inserted luxA and luxB
containing construct in transgenic N. tabacum corollas,indicating N. tabacum promoter-driven expression at differentdevelopmental stages (96).
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(102). Since that time, both luc and bacterial luxABassays have been used in environmental sampling.
Prosser et al. (1996) reviewed how luminometry andCCD image-enhanced microscopy may be useful indetecting naturally luminescent bacteria in environmen-tal samples, to monitor their growth and metabolism onsoil particles, microbial survival and recovery, microbialpredation, plant pathogenicity, rhizosphere colonizationand the reporting of gene expression in environmental
samples (103). They suggested that this technology mayalso be used to distinguish genetically modified bacteriafrom indigenous bacteria in environmental samples.
Using a mycobacteriophage T5–luc construct, Sarkiset al. (1995) infected Mycobacterium smegmatis toproduce luciferase expression both in the bacteria andin the post-infection lysogen (104). Such luciferasereporter phages may be used to test for the presence ofdrug-resistant or drug-sensitive M. smegmatis strains, aswell as for the rapid identification of other effectiveantimycobacterial agents.
Loessner et al. showed (1996, 1997) that the Listeriamonocytogenes-specific bacteriophage A511, whentransformed with the Vibrio harveyi luxAB gene, can bea sensitive detector of viable Listeria cells in environ-mental and food samples within 24 h, rather than theusual 3 days required for traditional culturing (105, 106).Also, competitive PCR and imaging analysis can be usedto quantify the number of luciferase gene copies insediment samples to which Synechocystis 6803-luc cellswere added (107). The authors suggest that this techniqueshould have applications for quantifying geneticallymodified cyanobacteria in nature.
Although not directly imaging luciferase expression,Unge et al. used a mini-transposon construct bearing agfp–luxAB fusion to simultaneously assay bacterial cellnumbers and populational metabolic activities in specificpopulations of E. coli DH5� and Pseudomonas fluor-escens SBW25 in soil samples (68). Bacterial numberswere determined by flow cytometric monitoring of GFP-expressing cells. LuxAB expression, as determined byluminometry, was shown to be dependent on nutrientlevels and hence metabolic activity.
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A bioluminescent reporter strain of Escherichia coli(O157:H7) containing the full bacterial luciferase(luxABCDE) operon was inoculated in buffer and infecal slurry, which were both placed on surfaces of beefcarcases to determine the interaction between potentialbacterial pathogens and human food animal tissues (108).A sensitive photon-counting camera was used tovisualize the presence of bacteria in real time. The fulllux operon in O157:H7 renders substrate additionunnecessary.
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Imaging of luciferase expression in plant and animal celllines has found more diverse applications than hasimaging of luciferase in bacteria. These applicationsinclude the imaging of protein site-specific secretion,protein trafficking and protein targeting to the imaging of
Figure 5. Visualization of bacterial luciferase expression inthe leaves (A) and fruit (B) sections of transgenic tomatoLycopersicon esculentum) plants (97, 172).
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transgenic promoter expression, real-time gene activa-tion, cell injury-induced expression and regulation, thedetermination of ATP and free Ca2� concentrations, andthe visualization of immune response.
Keller et al. showed in 1987 that a firefly luciferin–luciferase cDNA construct could be expressed in monkeykidney cells and that the gene product was targeted to theperoxisomes via a putative peroxisomal targeting proteintranslocation sequence (109). Using cDNAs of luc withthe peroxisomal targeting sequences, Gould et al. helpedto demonstrate that such sequences for protein transportare conserved from yeasts to plants, insects and mammals(110, 111). The expression of firefly luciferase (Luc) hasbeen imaged in single COS-7 cells by White et al. (1990)(112).
A method for photographic film detection of fireflyluciferase expression regulated by the simian virus 40promoter in mammalian cells was developed for use witha polyester mesh replica plating technique to determineluciferase expression in mammalian cells (113).
Site-specific protein secretion from transformed Chi-nese hamster ovary (CHO) cells, containing luciferasefrom the marine crustacean ostracod Vargula hilgendorfii(Luc), was visualized in real time using an image-intensifying system (114).
Transient expression of luciferase fusion proteins hasbeen an important tool in cell imaging. Luciferase cDNAfused to the 5�-flanking region of the rabbit collagenasegene containing a wild-type promoter showed anincreased expression after mechanical injury to thesmooth muscle Rb-1 cell line (115). In hormone studies,a modified luciferase expression system was used for real-time measuring of gene expression in endocrine cells(116). Jausons-Loffreda et al. (1994) used single photon-counting technology to document steroid hormoneactivity in transformed cell lines expressing fireflyluciferase, activated by chimeric constructs of the bindingdomain of the Gal4 yeast protein fused to the hormone-binding domains of various steroid receptors (117). Usingpromoter–luciferase reporter constructs, dynamic geneexpression was visualized in real time and quantified innursing rat lactotrope cells by Castano et al. (118), whoproposed that similar constructs could be used to visualizegene expression in any normal cell type. The Renillareniformis luciferase cDNA was expressed transiently insimian COS-7 cells and stably in murine C5 fibroblastsand in extracts (61). A firefly luciferase–aequorin fusionprotein was used in HeLa cells to detect rapid changes inATP and free Ca2� levels, based on light emission inresponse to C9 complement attack (119). Quantificationof ATP concentrations has also been carried out in ratcardiac myocytes by Dorr et al. (1989) (120).
Rat pituitary tumour cells (GH3) were transformedwith a construct containing the firefly luciferase gene and5000 bp of the 5� flanking region of the human PRL(prolactin) gene, subjected to luciferin, and then imaged
by CCD photon-counting for time periods up to 72 h(121). Basal PRL promoter–luciferase activity wascompared to stimulated activity after the addition ofsuch stimuli of the PRL promoter as thyrotropin-releasing hormone (TRH), forskolin, calcium channelagonist Bay K8644, and basic fibroblast growth factor(bFGF). Individual cells could be imaged.
Visualization of longer-term transient luciferase ex-pression in mammalian cells has also been possible.Various modified polylysine constructs with couplingreagent sulpho-LC SPDP were transfected with luciferaseinto HuH7 human hepatoma cells and imaged 2–16 daysafter transfection (122).
Real time-intensified CCD camera imaging of fireflyluciferase (Luc) expression has been utilized to confirmthat glucose may induce insulin gene transcriptionthrough increases in intracellular Ca2� concentration inMIN6 �-cells when glucose, insulin or the Ca2� channelinhibitor verapamil were added (Kennedy et al. 1999a)(123). Furthermore, Kennedy et al. (1999b) usedrecombinant firefly luciferases and photon-countingimaging to visualize concentration changes in free ATPin subdomains of single living MIN6 and primary �-cells(124). When control was made for pH, free ATP levelscould be visualized in real time in the cytosol, at theplasma membrane and in the mitochondrial matrix byusing luciferases specifically targeted to these threesubdomains (cLuc, pmLuc, and mLuc), respectively.This was an excellent example of subcellular imagingof luciferase expression.
Chinese hamster ovary (CHO) cells with stable expres-sion of both a CRE–luciferase reporter construct and thehuman pituitary adenylyl cyclase-activating peptide (PA-CAP) receptor were exposed to receptor agonists for each.Visualization of luciferase light emission, as well asfluorescence, was used to visualize Ca2� mobilization andthe induction of adenylyl cyclase activity (125).
Pancreatic �-cell activation of phosphatidylinositol 3�-kinase has been visualized by photon-counting imagingwith an intensified CCD camera, using intranuclearinjection of recombinant promoter fused to firefly andRenilla luciferase cDNAs (126). Elevated glucose levelsinduced pre-proinsulin (PPI) and liver-type pyruvatekinase (L-PK) promoters fused to firefly and Renillaluciferase cDNAs, thus producing light emission whichwas used to visualize single cells (127).
By constructing a less stable (and fainter) luciferasewith a shorter functional half-life and implanting it intohuman breast cancer T-47D cells under the control ofoestrogen response elements, Leclerc et al. (2000) wereable to observe real-time gene expression in single livinghuman breast cancer T-47D cells (128).
Increases in Ca2� concentrations in HeLa cells andskeletal myotube cells caused by agonist addition havebeen shown to raise levels of ATP production (129).Ainscow and Rutter (2001) have shown the significance
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of this by quantifying (via continuous photomultipliermonitoring and intensified CCD imaging of aequorin andfirefly luciferase) the expression introduced by adenoviralvectors in rat pancreatic islet (MIN6) cells and stimulatedby the addition of glucose (130). This further indicatedthat the expression of aequorin and Pluc is dependent onCa2� and ATP concentrations.
Synthetic esters of luciferin, a number of which weresubstrates for purified esterases, apparently hydrolysedinto luciferin in intact mammalian cells, where theycaused peaks in expression at levels six-fold higher thanwild-type luciferin (131). This suggests that such estersmay be used to assay for luciferase activity inmammalian cells where the concentration of luciferinwould be the rate-limiting factor.
Although imaging luciferase in individual plant celllines and cultures is more difficult, and therefore has notbeen done as often as in animal cell lines, several reportsare available. Polyadenylated luciferase mRNA electro-porated into tobacco protoplasts has been imaged byvideo at a wide range of levels of expression (132).
Another advance was the transformation of mouseembryonic stem (ES) cells with a cDNA constructexpressing the Ruc–GFP fusion protein, and its visual-ization (Fig. 6) (69).
Recently a gfp–luxABCDE reporter construct, underthe control of the XylA promoter, was used to monitor theexpression and temporal induction of the quorum-sensingaccessory gene regulator (agr) in S. aureus infectingbovine mammary epithelial MAC-T cells (133). The
reporter gene expression was occasioned by the virulencefactor-mediated escape of S. aureus from the hostendosome and its ensuing intracytoplasmic growth.
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A most exciting feature of luciferase expression imaging isthe ability to visualize gene expression in individual cellsin real time. Wood and DeLuca (1987) showed thatvectors can be checked for functional coding sequences byincluding a firefly luciferase gene probe, expressing it in E.coli, and detecting it by photographic film after theappropriate luciferin is added (134). Back in 1990, Hooperet al. reviewed low-light imaging with particular emphasison charge couple device (CCD) imaging of single cellsexpressing luciferase (135). Recombinant Vaccinia virusbearing firefly luciferase were added to cell culture.Virally transduced cells could be detected by imaging ofLuc expression at a level of one infected cell per million.Hooper et al. further suggested that imaging of luciferase-expressing viruses could be used to detect virus deletionmutants. Photon-counting CCD imaging of firefly lucifer-ase activity was used by Kennedy et al. to visualizeglucose L-pyruvate kinase (L-PK) promoter activity insingle living pancreatic islet �-cells at different glucoselevels (136). Detection of the L-PK promoter-driven fireflyluciferase activity was standardized using CMV promoter-controlled Renilla reniformis luciferase activity. Highintensity real-time photon-counting imaging was able to
Figure 6. Visualization of mouse embryonic stem (ES) cells transformed with a cDNA construct expressing the Ruc–GFP fusion protein (69). A and D are the light images; B and E are the images of green fluorescence under UV light. Cand F are overlays of A and B, D and E, respectively.
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detect firefly luciferase and aequorin reporter genesactivated by insulin-induced MAP kinase signalling insingle CHO.T cells, even when under the control of weakpromoters, according to the findings of Rutter et al. (1995)(137). Rutter et al. (1996) also used CCD camera imagingto visualize intramitochondrial Ca2� concentrations inCHO.T cells, using recombinant, Ca2�-sensitive aequorin(138). Regulation of human cytomegalovirus (hCMV) andhuman immunodeficiency virus (hHIV) gene expression inindividual, intact HeLa cells has been imaged usingconstructs with a firefly luciferase reporter gene down-stream of viral promoters (139).
Single bacterial cell microscopic imaging also is anemerging field. Hill et al. (1994) were the first to reportthe imaging of bioluminescence in individual bacterialcells (140). Using photon-counting, they were not onlyable to visualize individual bacteria (such as Pseudomo-nas fluorescens) that had been transformed with luxAB-bearing plasmids and transposons, but were also able toimage naturally luminescent Photobacterium phosphor-eum. Furthermore, they were able to show that expressionvaried over time, due to cell cycle-related changes inmetabolic activity.
Two low-light imaging systems were compared toassess their efficiency in visualization of single cells ofVibrio fischeri (MJ-1) and of a strain of Pseudomonasputida both of which were expressing V. harveyi luxAB(141). The authors found that a slow scan liquid N2
cooled CCD (C-CCD) camera was preferable for higherresolution of single cell signal at longer exposure times,but that a photon-counting CCD (PC-CCD) camera wasto be preferred for living cells at shorter exposure times,even though the resolution was somewhat lower. Phieferand colleagues (1999) quantified relative photon fluxfrom individual cells of Vibrio fischeri and V. harveyiusing photon-counting microscopy (142). Vibrio fischeriluciferase was found to be more stable in expression,while V. harveyi luciferase was found to be much morevariable in its light emission.
Even the rapid flashing of individual bioluminescentorganelles (scintillons), within individual dinoflagellatesof the species Pyrocystis noctiluca, has now beensuccessfully imaged using video image intensifier lightmicroscopy (143). The flashing, which occurs withfraction of a second rapidity, was induced by concen-trated citric acid stimulation.
In another review (1994), Hooper et al. summarized ingeneral the new improvements in low-light imagingtechnology. They discussed the hardware and softwareavailable, noted the rapid non-invasive advantages oflow-light imaging in reporter (luc, lux) gene expression,in intracellular expression and in analysis of tissuesections, as well as immunoassays, gels and blots (144).Also in 1994, Nicolas reviewed the varied applications inthe biological sciences of low-light photon-countingimaging, from the large-scale (immunoassays, DNA
probes and in vivo imaging of expression and promoteractivity) to the small scale (in situ hybridization andcellular luciferase expression) (145).
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In some ways, the goal of luciferase imaging has alwaysbeen to monitor processes in living, multicellularorganisms non-invasively.
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As in cell and tissue cultures, a very wide variety ofapplications have been made of luciferase expressionimaging in multicellular animals—both living and post-mortem.
In 1989, Hohn-Berlage et al. showed that ATP in intactbrain cryosections can be imaged using firefly luciferin–luciferase, and that lactate concentrations can be likewiseimaged using V. fischeri luciferase and lactate dehydro-genase (146) (see also Paschen, 1985) (147). Luciferin–luciferase assays can also be used to image distributionsand abundances of respiratory metabolites such asglucose, ATP, and lactate in tissue sections (148).
Following liposome-mediated transformation of luci-ferse expression vectors, luciferase expression has beenmonitored in normal and atherosclerotic external iliacrabbit arteries (149). Replication-deficient adenovirus-bearing firefly luciferase and �-galactosidase genes weresuccessfully used to test the comparative efficiencies oftransgenic gene delivery to cultured Sprague–Dawley ratthoracic artery and aortic artery smooth muscle tissuecultures (150). Luminometer readings confirmed theluciferase assay differential.
In 1993, Mueller-Klieser and Walenta showed thespatial distribution and concentration of respiratorymetabolites (ATP, glucose and lactate) in rapidly frozentissue in absolute unit concentrations and at single-celllevel resolution, using photon-counting visualization ofluciferase expression coupled with any particular enzymeof interest (151). Single-photon count imaging ofluciferase assays was used to spatially quantify thedistribution of respiratory metabolites (ATP, glucose, andlactate) in cryosections of tumours and normal tissue(152). Luciferase light emission was proportional tometabolite concentration. These results were confirmedusing other metabolite quantification methods.
Rembold et al. (1997) used a replication-deficientadenoviral vector carrying an apo-aequorin cDNA with asarcoplasmic reticulum (SR) targeting sequence to infectintact rat tail arteries (153). In this way the authors wereable to measure fluctuation in the presence of free Ca2� inthe SR in the presence of coelenterazine, the apo-aequorin luciferin substrate.
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Using a luciferase reporter, Thierry et al. (1997)showed that when a lipopolyamine, a neutral lipid and aplasmid DNA are associated in the formation of lamellarvesicles, they can be used to compare in vitro and in vivotransfection efficiencies in mouse tissues (154). Theselamellar vesicle–DNA complexes have a higher in vitrotransfection efficiency than that of previously reportedliposome transgenic delivery systems.
Making use of the fact that muscles, injected with apromoter–firefly luciferase cDNA fusion construct, retainLuc activity for up to 60 days, Davis et al. (1997) showedthat, when a luciferase construct was co-injected with ahepatitis B virus surface antigen (HBsAg)-expressingDNA, the luciferase expression time is shortened to about5 days of strong expression and a cut-off after 20 days(155). These findings suggest that luciferase can be usedto image indirectly the progress of immune-mediateddestruction of muscle myofibre tissue.
The subcutaneous injection of a lipid gadoliniumcontrast complex containing Luc was imaged by Wisneret al. (1997) using MRI (156). The Luc was also imaged.The successful insertion of Luc with the complex into thecell provided a possible method for the visualizing of atransfection event. MRI imaging provided an indepen-dent check on the luciferase imaging.
In 1997, Contag et al. successfully monitored in vivotransient expression of SV40 promoter/enhancer–Lucfusion constructs, using intensified CCD imaging on thelungs of neonatal rats (157). Cationic liposome deliveryof the vectors was used. The expression vector was alsoinduced in mice and in human T-cells.
Endogenous ATP was measured simultaneously withthe release of acetylcholine from the isolated superiorcervical ganglion of a rat using firefly luciferin–luciferase(158). Using imaging of luciferase light emission, Marchet al. (1999) showed the feasibility of catheter-basedpericardial local delivery of adenoviral vectors for genetherapy in dogs (159). One of the recombinant adenoviralexpression vectors encoded luciferase in the cytoplasm.This was visualized post mortem in pericardial tissues insacrificed animals.
Similar imaging precision has been possible in livinganimals. In a landmark study by Edinger et al. (1999),HeLa cells stably expressing firefly luciferase wereintroduced via subcutaneous, intraperitoneal and intra-venous injection into SCID mice (160). Tumour cellkinetics and growth were monitored by whole-bodyphoton-counting visualization. Immediately postinjec-tion, the following tumour cell numbers could beobserved by low-light imaging: 1 � 103 cells in theperitoneal cavity; 1 � 104 cells at subcutaneous sites; and1 � 106 circulating cells.
Using a bispecific antibody, Reynolds et al. (2000)targeted an adenoviral vector encoding firefly luciferaseto the pulmonary endothelium of rats (161). Targeting ledto a 20-fold increase in target area luciferase expression
and a reduction in expression in non-targeted organs.Orson et al. (2000) imaged luciferase expressionlocalized in mice lungs by using an intravenouslyinjected, artificial, lung-targeting macroaggregated poly-ethyleneimine–albumin protein conjugates binding afirefly luciferase cDNA (162).
Sugihara et al. (2000) transfected the chloramphenicolacetyltransferase (CAT) gene and firefly luciferase (luc)expression cassettes by electroporation into the testiclesof living chickens (163). The authors were able to imageluciferase expression in and around the injection site. Aself-replicating sequence of the Epstein–Barr virus wasadded to stabilize luc expression in vivo.
Using a tetracycline-inducible promoter construct,Hasan et al. (2001) have shown that transgenic fireflyluciferase is suitable as a visualizing marker to monitorinduction of the expression of a second transgene inliving mice, in this case, Cre recombinase (164).
Recently, Lipshutz et al. (2001) tested prenatal, inutero delivery of recombinant adeno-associated virus(rAAV) vectors carrying firefly luciferase via intraper-itoneal injection in mice (165). Luciferase expressionwas visualized by whole-body imaging in all injectedanimals. At birth, the highest Luc expression was in theperitoneum and liver, with lower expression levels in theheart, brain and lung. Expression persisted for as long as18 months in the peritoneum. No antibodies against Lucor rAAV were detected and no liver cell damage wasreported. These data suggest that in utero DNA deliveryis a safe and effective method of prenatal gene therapy inanimal models.
Muramatsu et al. (2001) have demonstrated nutrition-ally-regulated transgene expression in mouse liver usinga liver-type phosphoenolpyruvate carboxykinase(PEPCK) gene promoter driving firefly luciferase ex-pression (166). Fasting induced a PEPCK-driven 13-foldincrease of luciferase expression in the liver, but nosimilar induction was found in muscle tissue for either thePEPCK promoter or a control SV40 promoter.
Yu et al. used the ‘Gene Switch’ progesteroneantagonist (RU486)-inducible system co-transformedwith a Renilla luciferase–gfp fusion construct (CMV–ruc–gfp) to visualize inducible gene expression in COScell culture, and also intramuscularly in male nu/nu mice(167). RU486-induced Ruc–GFP expression was visua-lized using low-light imaging.
In short, numerous studies have shown the efficacy ofluciferase imaging in modelling and observing complexgene activation events taking place in vivo in live animalsand in tissue sections.
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In many ways, the ultimate goal of luciferase imaging is
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not only to monitor gene expression non-invasively andin real time in living, multicellular organisms, but also toreveal and document spatial, tissue and cell type-specificexpression in genetically altered (transgenic) organisms.Such applications of luciferase imaging have advancedrapidly especially in plants. By 1994, Langridge et al. hadaddressed the usefulness and convenience of using thebacterial luciferase (luxAB) system in a variety ofeukaryotic transgenic organisms and reporter geneapplications (97).
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Global imaging of luciferase-expressing transgenicplants has served further research in many geneexpression studies, from simple expression of luciferasesin transgenic plants, to regulation of developmental genesto the gene expression of wound response, and to thedetection of expression in response to thermo-osmoticstress. Photographic film was used by Ow et al. (1986) tovisualize transient and stable firefly luciferase (Pluc)expression in Nicotiana tabacum (168). In 1986, Konczand Schell imaged tissue-specific chimaeric LuxA andLuxB expression in Daucus carota and Nicotianatabacum (169). By 1987, Koncz et al. (170) haddemonstrated the successful assembly and expression ofLuxA and LuxB from Vibrio harveyi in transgenic carrot(Daucus carota) and tobacco (Nicotiana tabacum) plantsvia Agrobacterium-mediated T-DNA or direct DNAtransformation. Furthermore, Koncz et al. (1990) dis-cussed the advantages and disadvantages of expressingbacterial (Lux) and firefly (Luc) luciferases as reportergenes in transgenic plants (171).
A promoterless luxA gene was fused to the 5� end of aT-DNA, adjacent to a cauliflower mosaic virus 35Spromoter-driven selectable marker, and inserted intotobacco leaf explants to generate transgenic tobaccoplants. Some of these transformed plants were found, bylow-light imaging, to express luciferase in only oneorgan, e.g. a floral corolla. This system thus served as apromoter search assay to find organ-specific promoters(172, cf. 96) (Fig. 7a, b). Sequencing of linked genomicDNA from these plants allowed for the isolation ofdevelopmental genes and their regulatory elements.
By 1994, Mayerhofer et al. had visualized stableRenilla luciferase expression in transgenic tobaccoleaves, tomato fruit, and potato tubers (173). In afollow-up study, Mayerhofer et al. (1995) used Agro-bacterium-mediated transfection to create transgenicalfalfa (Medicago sativa), tomato (Lycopersicon escu-lentum), tobacco (Nicotiana tabacum) and potato (Sola-num tuberosum) plants (Fig. 8) with high levels ofRenilla luciferase (Ruc) (60). The authors found that Rucexpression levels are substantially higher than that ofeither firefly (luc) or bacterial (Lux) luciferase.
Firefly luciferase (luc) was fused with an Arabidopsiscircadian regulator, cab2 (chlorophyll binding protein 2)promoter, and the activity of Cab2 was inferred byvisualizing luc expression both spatially and temporallyin seedlings by low-light video-imaging (1992) (174). Aluc fusion construct carried by transgenic Arabidopsisplants was later (1995) used to identify plants withmutant long- and short-period circadian cycle genotypes,namely plants with mutations in toc1, a gene involved inthe timing of Cab regulation (175). Another group (1998)used luciferase Cab promoter–luciferase fusion vectorsand video-imaging to visualize transgenic expression in
Figure 7. Visualization of the expression levels of abidirectional mas P1,P2 promoter–luxA and luxB gene constructwithin specific tissues of transformed N. tabacum, such as (A)in flowers (non-transformed flower image on the left) and (B) inleaves. Placement of transgene accomplished via Agrobac-terium-mediated T-DNA or direct DNA transformation (172).
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tobacco seedlings, to determine how the photoreceptorphytochrome circadian oscillator regulates expression ofcab genes, which drive early seedling development (176).Recently, Schutz and Furuya (2001) monitored Cabsignalling in the cotyledons of Nicotiana tabacum usingcab-luciferase reporter genes (177).
Luciferase expression in transgenic plants has beenused to evaluate imaging equipment and technology.Mutants of transgenic seedlings of Arabidopsis thaliana,containing native promoter-inserted luciferase con-structs, were monitored by low-light imaging (178).When compared, they found that a cooled CCD camerawas more efficient than an intensified CCD camera indetecting mutants in the screen.
Firefly luciferase (luc) under the control of the stress-responsive RD29A promoter was introduced into Arabi-dopsis plants by Ishitani et al. (179, 180). Induction of theendogenous RD29A gene was visualized using highthroughput in vivo luminescence imaging. Under differ-ent conditions, expression of the various stress/osmoticresponse pathways was monitored. These pathwaysincluded those that are both phytohormone abscisic acid(ABA)-dependent and ABA-independent. Further workby Ishitani et al. (1998) established that inducibleluciferase expression in Arabidopsis could be used tofind a temperature cold-response gene (HOS-1) mutant(181).
The general utility of imaging luciferase reporters intransgenic plants has been amply demonstrated, not onlyfor eukaryotic Luc but also for bacterial Lux. Langridgeand Szalay (1998) used low-light intensified photon-counting imaging to visualize bacterial luciferase (Vibrioharveyi) and eukaryotic luciferase (Renilla reniformis)
light emissions as markers for transformation andreporters of gene expression in transgenic Arabidopsisthaliana and Nicotiana tabacum. Both lux and ruc servedas promoter search reporter genes (182). Langridge et al.have also imaged bacterial luciferase expression in thetomato, Lycopersicon esculentum (see Fig. 5a, b), and inthe potato, Solanum tuberosum (97).
The success of luciferase expression in plants has beenextended to unicellular algae. Anthozoan Renilla reni-formis luciferase (Ruc) has been successfully expressedin the chloroplast of the alga Chlamydomonas reinhardtii(183). A cryogenic CCD camera was used to image thelight-emitting transgenic algal colonies.
Firefly luciferase (Luc) imaging has been used inevolutionary studies in Arabidopsis. Jelesko et al. (1999)documented unequal meiotic crossing over in Arabidop-sis thaliana plants with a synthetic rbcsb gene clustercomposed of �rbcs1b::luc–rbcs2b–rbcs3b, instead of thewild-type rbcs1b–rbcs2b–rbcs3b (184). Over 1 millionF2 generation seedlings screened by low-light photon-counting imaging yielded three light-emitting seedlingswith a chimeric �rbcs1b::luc–rbcs3b gene cluster,expressing luciferase and a predicted rbcs26 duplication.These results were confirmed by molecular methods.Luciferase imaging was thus used to assay directly thefrequency of evolutionary gene conversion in A. thaliana(�3 � 10�6).
Xiong et al. (1999a, b) utilized an efficient method ofhigh throughput genetic screening of hormone andenvironmental stress signal transduction mutants ofArabidopsis thaliana, using the firefly luciferase (luc)gene regulated by a cold, osmotic stress and ABA-responsive promoter (185, 186). A thermoelectrically-cooled CCD camera was used to image the plants underaddition of ABA and change of temperature conditions.The system allowed screening and recognition of high-and low-expression mutants.
Luciferase imaging, in addition to being used tovisualize plant promoter–gene response to thermo-osmotic pressures, has also been used to visualize plantgene response to infection. In 1996, Giacomin and Szalayutilized Pseudomonas infection of Arabidopsis thalianato induce expression of the phenylalanine ammonia-lyase(PAL1) promoter fused to the luxF gene (187). In anotherstudy, transgenic Arabidopsis carrying a gst1::luctransgene were used to image the spatial and temporalconcentrations of reactive oxygen intermediates (ROIs)in response to an assault by infectious Pseudomonassyringae pv. tomato (188) (for more details, see thesection on Imaging of host–pathogen interactions,below).
Transgenic seedlings were engineered by Urwin et al.(2000) to carry a bicistronic gene with both GFP andfirefly luciferase ORFs linked by the encephalomyocar-ditis (ECM) IRES element and regulated by the CaMV35S promoter (189). Both GFP and Luc were expressed
Figure 8. Visualization of Renilla luciferase gene expressionin transformed potato tubers (Solanum tuberosum) (cf. 60, 173).
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and detected by in vivo imaging, indicating that the ECMIRES element facilitates the translation of the secondORF in vivo. Northern blot analysis also confirmed thepresence of both GFP and luciferase products.
Transgenic tobacco plants (Nicotiana tobacum L.)expressing firefly luciferase (luc) driven by the Arabi-dopsis phenylalanine ammonia lyase 1 (PAL1) promoterhave been imaged in vivo for up to 24 h after the additionof exogenous luciferin (190). Wounding enhanced the lucexpression in these plants, suggesting that there arebarriers in plants to ordinary luciferin uptake intransgenic luciferase assays.
Using a fusion of the GA5 promoter (growthphytohormone gibberellin) and firefly luciferase (ga5–luc) cDNA, Meier et al. (2001) used imaging to show thatGA5 promoter regulation of GA occurs at the level oftranscription (191). Imaging allowed the investigators toidentify recessive mutants with high Pluc expression.
Recently, van Leeuwen and colleagues (2001), usingthe CaMV 35S, modified CaMV 35S and the Arabidopsisthaliana lipid transfer protein gene promoters inconjunction with the firefly luciferase (luc) gene, wereable to show that variant levels of transgene promoteractivation result not only from the integration site(position effect) but also from spatial and temporalpromoter regulation (192). These patterns are inheritedby the next generation. Expression levels were monitoredin leaves of individual transgenic plants during a 50 dayperiod, both by imaging and by assaying local mRNAlevels. Further work has shown that matrix-associatedregions (MAR) elements in proximity to the transgenecause a varying effect on the temporal regulation of thetransgene expression between individual plant transfor-mants (193).
In summary, luciferase imaging of transgenic plantshas thus been used in the study of whole plant geneexpression and regulation, as well as in the screening ofmutants.
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Whole-body imaging of luciferase expression in trans-genic animals has not proceeded as rapidly as in plants,largely because of the greater difficulty of generatingtransgenic animals. However, during the past decade,significant work has been carried out, particularly inimaging of gene expression during development intransgenic embryos.
In 1990, Tamiya et al. first imaged the distribution offirefly luciferase expression in transgenic zebrafish(Brachydanio rerio) (194). Zebrafish have been afavourite transgenic model because zebrafish eggs areeasily accessible to DNA injection. Mayerhofer et al.visualized the spatial distribution of firefly luciferaseexpression in transgenic zebrafish using low-light video-image analysis (195).
Using photon-counting imaging, Matsumoto et al.(1994) found that mouse embryos from transgenicparents carrying the paternally inherited chicken �-actinpromoter–luc construct emitted detectable light at thetwo-cell stage (196). Luciferase mRNA was found evenat the one-cell stage. Thus, luciferase imaging helped todemonstrate the very early onset of embryonic geneexpression.
Expression of secreted Vargula luciferase (Vuc) wasimaged in live pre-implantation mouse embryos fromhomozygous transgenic mice containing luc and vuccDNAs, using image intensifiers connected to a CCDcamera (197). In this way, developmental gene expres-sion modulation could be observed and assessed inindividual embryos by two different luciferases.
Transgenic mice (adults and neonates) with HIV LTR-luc (firefly luciferase) constructs were found to expressluc luminescence after intraperitoneal, topical absorption,or topical electroporative delivery of D-luciferin substratein DMSO (198). Both near the surface and deeper,visualization of luc expression was possible using inten-sified CCD (ICCD) and cooled CCD (cCCD) imaging.
Transgene integration efficiency was determined bybioluminescent visualization screening in microinjectedbovine embryos (199). A murine HSP70.1 promoter waslinked to a firefly luciferase (luc) cDNA and micro-injected into zygote pronuclei produced in vitro. Plucexpression was then visualized in the resulting embryos.
Various promoter/enhancer–Pluc constructs weremicroinjected into pre-implantation bovine embryosand their expression assayed by luminometer andphoton-imaging at 2 and 6 days postinjection (200).These experiments tended to document the persistence ofearly somatic cell promoter activation during embryonicdevelopment.
Utilizing transgenic mice that carry an integratedmurine heme-oxygenase 1 (HO-1)-luc cistron, Zhang etal. (1999) were able to visualize levels of tissueoxygenation in real time using intensified CCD cameraimaging (201). The changing O2 concentrations weretriggered by intraperitoneal injections of CdCl2. Bothhypoxic and hyperoxic tissue conditions altered HO-1promoted luciferase expression.
Taking advantage of the recently discovered fact thatthe Cytomegalovirus immediate–early gene 1 (CMV IE-1) enhancer–promoter is selectively expressed in onlycertain brain cells, Sigworth et al. imaged Pluc expres-sion in brain sections from two lines of transgenic C3H/B6 mice, using a liquid nitrogen-cooled CCD camera(202). One line of mice contained the human CMV::lucfirefly luciferase construct and the other contained thehuman c-fos::luc firefly luciferase construct. TheCMV::luc mice brain sections maintained at 30C or36C showed discrete expression patterns, especially inthe dorsal suprachiasmatic nucleus circadian pacemakerof the hypothalmus.
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Making use of an E1-deletion adenovirus expressingPluc under the control of a cytomegalovirus promoter,Wu et al. (2001), utilizing cooled CCD imaging, wereable to visualize the location, magnitude and persistenceof Pluc expression in Swiss Webster mice (203).
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Using photoproteins such as luciferase and GFP to imagethe progression of tumour growth in vivo is an importantand rapidly advancing field (204–206). We summarizeselected papers published recently. Contag et al. (2000)have discussed the use of GFP and firefly luciferase asreporter in real time, in vivo imaging of tumours. Theyalso discussed the use of these reporter genes in studyingcellular and molecular aspects of neoplastic disease,growth and regression under therapy (207). The authorsshow that photon imaging is sensitive enough now todetect 1000 luciferase-labelled tumour cells spreadthroughout a mouse peritoneal cavity. The authors furthernote that, while GFP is adequate for high-resolutionanalyses after tumour localization in vivo, luciferase-labelling is superior in tracing the progress of neoplasticgrowth from a few cells to extensive metastases. Infuture, luciferase reporter monitoring of cancer genetherapy is a suggested application.
In a recent tumour imaging study, Rehemtulla et al.(2000) used 9L rat gliosarcoma cells stably transfectedwith firefly luciferase (9LLuc) to produce orthotopic braintumors (208). Other luciferase-transformed tumour lineshave been used in intraperitoneal, subcutaneous andintravascular models to visualize the kinetics of tumourgrowth and response to therapy. Cooled CCD camera andmagnetic resonance imaging (MRI) showed an excellent0.91 correspondence between imaged photon emissionand MRI-measured tumour volume. In evaluatingchemotherapeutic treatment modalities, CCD and MRIconfirmed each other to a p = 0.951 confidence level. It issignificant and promising that luciferase in vivo imagingcompared so favourably with MRI as a tool for assessingthe spatial extent of in vivo tumours.
In a recent important luciferase imaging study,Honigman et al. tested a number of parameters of invivo imaging in mice and rats (209). After injectingvarious plasmid vectors, recombinant viruses and trans-fected tumour cell-lines (see Table 2), the authors imagedbladder, bone, dermis, liver, muscle, peritoneum, pros-tate, salivary glands, teeth and testis in mice and rats.They used visualization of Luc expression to check organspecificity, efficiency of substrate delivery, long-termmonitoring of tumour growth, promoter specificity, andefficiency of injection methods using image-intensified(ICCD) and cooled (CCCD) charge-couple devicecameras. Location, magnitude and duration of Luc
expression were simply and reproducibly determined byCCCD photon-counting methods. Luminometry servedto monitor Luc activity within organ and cell extracts.
In spite of the remarkable progress made, much moreremains to be done with luciferase visualization oftumours in vivo. Tumour imaging in general is animportant interface between basic research and theclinical applications. Luciferase imaging promises asignificant role in this burgeoning field, perhaps even-tually in the visualization of tumours in humans.
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Many of the marine bioluminescent organisms are foundto exist in symbiotic metazoan–bacterial interactions andso are involved in natural bioluminescent in vivo‘visualization’ (3). However, most of the applied scientificuses of luciferase imaging directed at such interactionshave tended to study host–pathogen relationships.
One of the earliest and most exciting areas of luciferasein vivo imaging has been the real-time visualization ofbacterial infection in living organisms, both plants andanimals. Back in 1986, Legocki and co-workers usedtransformed Bradyrhizobium japonicum carrying luxAB(V. harveyi) under the control of the B. japonicumnitrogenase nifD promoter to monitor the presence of N-fixing bacteria in soybean root nodules, and so indirectlyto visualize N-fixation (Glycine max var. Wilkin) (210).Cell extracts of root nodules grown on plants without N2
were assayed for LuxAB activity by luminometer,indicating nif-driven LuxAB synthesis. LuxAB-trans-formed E. coli bacteria were used as a negative control.LuxAB fusion expression in transformed B. japonicum-infected soybean root nodules was successfully visual-ized using photographic film (Fig. 9a, b) and low-lightintensified video microscopy (211). Using N. tabacumtransformed with an auxin-stimulated bidirectional maspromoter–luxAB gene fusion construct, it was possible tovisualize the spread of Pseudomonas syringiae infectionin N. tabacum leaves (Fig. 10) and of Agrobacterium-induced crown gall in N. tabacum stem sections (Fig. 11)(97).
In 1993 Wang et al. imaged the expression of luxAB-bearing Bacillus thuringiensis within sixth instar larvaeof Manduca sextans (tobacco hornworm) 10 min post-injection and also after larval ingestion of the bacteria(Figure 3a, b) (95). The B. thuringiensis bacteria weretransformed with a plasmid containing luxAB under thecontrol of the XylA promoter (B. megaterium). Arecombinant baculovirus (polyhedrin promoter–luc) wasdeveloped for use as a rapid luminescent plaque assay tooptimize concentrations of recombinant baculoviralinfection in insect cell cultures and larvae (93).
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64 REVIEW L. F. Greer and A. A. Szalay
Figure 9. (A) Visualization of transformed Bradyrhizobium-infected soy-bean plant cells (Glycine max) in cross-sections of root nodules through LuxABlight emission (211). (B) Visualization of light emission in root nodules ofsoybean plants Glycine max grown in the absence of N2 in the gross medium.The bacterial inoculant was Bradyrhizobium japonicum, stably transformedwith the nifD promoter–luxAB fusion gene construct (211, cf. 210).
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Figure 10. Visualization of the spread of pathogens, Pseudomonas syringiae, in N. tabacum carrying the bidirectionalmas promoter-luxAB gene fusion construct (97, 170).
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66 REVIEW L. F. Greer and A. A. Szalay
Expression of Pluc within invading recombinantvaccinia virus has been imaged in African green monkeyBCS-40 kidney cells (212). Time exposure with sensitivefilm was used to capture the images of the light-emittingviral plaques in cell culture. Although not imaged, therecombinant luc-bearing vaccinia virus could be assayedin target organs in BALB/c mice.
Luciferase expression in transgenic Arabidopsis thali-ana transformed with a phenylalanine ammonia-lyasepromoter (PAL1)–luxF fusion was used to imagelocalized activation of PAL1 by infection of Pseudomo-nas syringae pathovar tomato, using photon counting(187). The PAL gene encodes phenylalanine ammonialyase, which catalyses the first step in a plant develop-ment pathway responding to environmental stresses, suchas infectious invasion.
In 1995, Contag et al. showed that bacterial infectionscould be traced non-invasively in vivo in mice usingStaphylococcus typhimurium transformed with a vectorcarrying constitutively expressed bacterial luciferase(lux) (213). Recently, Francis et al. (2000) showed thatStaphylococcus aureus transformed with the rearrangedPhotorhabdus luminescens lux operon (luxABCDE) can
be used to visualize the presence of infection and theeffectiveness of antibiotic treatment by direct whole-body imaging of mice after intramuscular injection of therecombinant bacteria (214). A similar method forStreptococcus pneumoniae has since been developed byFrancis et al. (2001), using a Gram-negative transposonbearing the luxABCDE and the kanamycin resistancegene (kanr) in one promoterless operon allowingtransformed, luminescent, kanamycin-resistant bacteriato be non-invasively visualized in vivo in murine models(215). Rocchetta et al. (2000) have used clinical E. coliEC14 transformed with the P. luminescens lux operon tovisualize bacterial infection in rat thigh muscles, using anintensified CCD camera system (ICCD) (216). Theimaging system was sensitive enough to achieve goodstatistical correlation between luminescence and viablebacterial cell numbers, both with and without thepresence of antimicrobial agents. Rocchetta et al. proposethe use of this technology for in vivo, therapeutic testingof antimicrobial agents.
Viral infection has also been visualized by luciferaseimaging. By using recombinant herpes/pseudorabiesvirus bearing luc, it is possible with ultra-high-sensitivity
Figure 11. Visualization of the auxin-activation of the bidirectional mas promoter-luxAB fusion construct in the stemsections of transgenic N. tabacum, which carry a crown gall tumour caused by the wild type Agrobacterium tumefaciens,strain C58 (93).
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photon-counting enhanced video imaging to visualize inreal-time the progress of viral infection and destruction ofmammalian cells in cell culture, even down to single cellresolution, according to Mettenleiter and Grawe (1996)(217) who used recombinant herpes/pseudorabies virus inAfrican green monkey kidney (VERO) cells. Theysuggest that in vivo monitoring of the spread of viralinfection in a living animal is now feasible. This has infact been done in insects. Langridge et al. (1996)successfully imaged not only recombinant baculovirusexpression of firefly luciferase in insect cell culture(Trichoplusia ni) 386 cells and Bombyx mori N-4 and SF9 cells, but also tracked in vivo the progress of a Pluc-transformed baculovirus infection in cabbage looperlarvae (Trichoplusia ni), using low light photon-countingvideo imaging (218).
Digital imaging microscopy has also been used (in2001) to determine the difference in transductionefficiencies on human A549 cells between two recombi-nant adenoviral vectors, AdCMVLuc and Ad5LucRGD(219).
Another recent development has been the direct in vivoimaging of bioluminescent CD4� T cells in a murinemodel (220). In a murine analogue of multiple sclerosis,experimentally-induced autoimmune encephalomyelitis,T lymphocytes transduced to express luciferase by pGCretroviral vectors were visually tracked by low-lightimaging cameras into the central nervous system. Long-
term transgene expression in the central nervous systemwas confirmed by histology.
Recent work has shown that injected bacterial infec-tions of Vibrio, Salmonella and E. coli transformed withan expression construct containing the P. luminescensluxCDABE operon can be visualized over many days inC57 mice (Fig. 12) and Sprague–Dawley rats (221). Low-light imaging was used to visualize bacterial infections inmuscles, in specific organs such as the kidney and liver invivo, and in excised hearts. Luminescent bacteria couldalso be observed through the mouse skull in vivo andthrough excised rat tibia.
Recombinant vaccinia virus (rVV-RG, LIVP strain),bearing a Renilla luciferase–gfp fusion gene under thecontrol of the vaccinia strong synthetic early–latepromoter (rVV–PE/L–ruc–gfp), have been used to imagerVV infection in CV-1 African green monkey kidneycells and in athymic nu/nu mice (222).
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Natural in vivo luciferase ‘visualization’ has taken place inmarine organisms since time immemorial in the oceans ofthe world. Scientific instrumental imaging of luciferaseexpression in living cells, tissues and organisms has madesignificant advances over the last few decades. Thisprogress was only possible because of fundamental
Figure 12. Visualization of intramuscularly-injected bacteria carrying the complete luxCDABE operon from P.luminescens in a C57 mouse (221).
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68 REVIEW L. F. Greer and A. A. Szalay
research. The mechanisms of the luciferin–luciferasebioluminescence systems are being elucidated and theirphylogenetic relationships are being worked out. Variousluciferase genes and cDNAs have been isolated andcloned. Advancement in visualization of luciferaseexpression in individual cells, in somatic plant and animaltissues and in transgenic plants and animals has been madepossible because of: (a) recombinant DNA construction ofvarious promoter–luciferase gene constructs and fusiongene products; (b) more efficient and precise delivery oftransgenic DNA and exogenous substrate; and (c)development of highly sensitive and versatile imagingtechnologies. Luciferase has been a useful reporter genefor imaging singly and in concert with other photoproteins,notably green fluorescence protein (GFP). Luciferaseimaging has also begun to play a crucial role in imagingof tumours and metastases. Other possible applicationsinclude the imaging of LRET between luciferases andGFPs in documentation of intracellular protein–proteininteractions. It seems likely that the most significant stepforward, although still in its infancy, is the ability ofluciferase imaging technology to resolve real-time geneexpression in individual cells. Perhaps the imaging of real-time gene expression within individual cells within livingorganisms may be a wave of the future.
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One chimaeric dream of modern science has been toobserve the world without disturbing it. In a small way, invivo, non-invasive, real-time imaging of luciferaseexpression and light emission in living cells andorganisms is at least in the spirit of that dream.
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We would like to thank former and present members ofthe Szalay laboratory, some of whose unpublishedimaging data we have included here; Dr Phil Hill forproviding us with a pre-publication manuscript on thework in his laboratory with intracellular infections ofStaphylococcus aureus; and various members of theSzalay laboratory for their help during the preparation ofthe manuscript, as well as Dr Bruce Wilcox, Dr WilliamLangridge, Dr William Hayes and others who madehelpful suggestions. LG was the recipient of a graduateresearch assistantship from the Department of NaturalSciences, Loma Linda University. The imaging researchin A. A. Szalay’s laboratory was supported by differentgrants cited in earlier publications. The present researchand unpublished images were supported with the help offunding by BSRG research grants provided by LomaLinda University to A. Szalay. We apologize to theauthors whose papers we were not able to include due toour own limitations.
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