Post on 07-Jul-2020
RealReal--time Reporter Gene Assaytime Reporter Gene Assay
Progress of realProgress of real--time reporter assay time reporter assay
using bioluminescence protein gene.using bioluminescence protein gene.
1. Typical reporter protein in Japan. 1. Typical reporter protein in Japan. 2. Real-time reporter gene assay by photon counting system.
3. Real-time reporter gene assay by imaging system.
Feature of Bioluminescence Reporter Assay
Aequorea aequoreaPhoto: Dr. Osamu Shimomura
Luciola lateralis
Valugula hilugendorfii
Firefly
Photo: Dr. K. Niwa
Photo: Dr. K. Ogho
GFP
Luciferin
Oxidation of Luciferin & Coelenterazine
620nm 560nm
+CO2+hν
O2
Renilla Luciferase
Coelenterazine Coelenteramide
Provided by Dr.H.Akiyama
Renilla Luciferase
Promoter A
Promoter B
Luciferase
D-Luciferin Coelenterazine
Dual Reporter Assay
Cell
Lysis of cells Luciferase
Activity
Renilla LuciferaseActivity
Promoter 2 Orange-Luciferase
Promoter 1 Red-Luciferase
MultiMulti--Color Reporter AssayColor Reporter Assay
Green-LuciferasePromoter 3
Cell
Lysis of Cells
D-Luciferin measurement of
Luminescence Intensity
Red
Orange
Green
Culture
SampleSample
Optical filter unitOptical filter unit
Advanced Method of Advanced Method of
Quantitative Color DetectionQuantitative Color Detection
PMTPMT
Optical filter unitOptical filter unit
F0F0
Relative Counts (F0) = G+O+R
SampleSample
Optical filter unitOptical filter unit
Advanced Method of Advanced Method of
Quantitative Color DetectionQuantitative Color Detection
PMTPMT
Optical filter unitOptical filter unit
F1F1 Relative Counts (F1) =
Κf 1g××××GGGG+Κf 1o××××OOOO+Κf 1r ××××R
ΚΚf1g : Transmittance of green luminescence off1g : Transmittance of green luminescence of f1 filter f1 filter
SampleSample
Advanced Method of Advanced Method of
Quantitative Color DetectionQuantitative Color Detection
PMTPMT
Optical filter unitOptical filter unit
F0F0 F2F2
Relative Counts (F2) =
Κf 2g××××GGGG+Κf 2o××××OOOO+Κf 2r ××××R
SampleSample
11 11 11F0F0 GG
Advanced Method of Advanced Method of
Quantitative Color DetectionQuantitative Color Detection
PMTPMTG, O, RG, O, R : Each color light: Each color light
11 11 11
κκf2gf2g κκf2of2o κκf2rf2r
κκf1gf1g κκf1of1o κκf1rf1r==F1F1
F0F0
F2F2
OO
GG
RR
F : F : Measurement valueMeasurement value
κκ : Transmittance of each color : Transmittance of each color
light of each optical filterlight of each optical filter
Optical filter unitOptical filter unit
F0F0 F1F1 F2F2
Endogenous Endogenous
genegene
RealReal--Time Reporter AssayTime Reporter Assay
(Live Cell Reporter Assay)(Live Cell Reporter Assay)
Living CellLiving CellLuciferase geneLuciferase genePromoterPromoter
Reporter vectorReporter vector
LuciferinLuciferin genegene
Continuous and realContinuous and real--time time
measurement of lightmeasurement of light
Inte
nsi
ty
Time
mRNAmRNA
LuciferaseLuciferase
LuciferinLuciferin
(in the culture medium)(in the culture medium)
Luminometer Luminometer
for realfor real--time reporter assay in living celltime reporter assay in living cell
Kronos (ABKronos (AB--2500)2500)
35mm Culture Dishes35mm Culture Dishes35mm Culture Dishes35mm Culture Dishes
on Turntableon Turntable
Keeping Constant Keeping Constant
Temperature (20Temperature (20--4545℃℃℃℃℃℃℃℃))
PhotomultiplierPhotomultiplier
TubeTube
Temperature StabilityTemperature Stability
303540
Temp(℃)
Dish temp
Setting on the new dish
Sensor in dish
Sensor in kronos
10152025
2006/12/2612:00:00 2006/12/2812:00:00 2006/12/3012:00:00 2007/01/0112:00:00 2007/01/0312:00:00 2007/01/0512:00:00Time and Date
Temp(℃)
Room temp
35.0
35.5
36.0
36.5
37.0
37.5
38.0
0 60 120 180 240 300 360 420
Time(sec)
Temp(℃
)
Preset Temperature : 37℃
COCO22 ConcentrationConcentration
6 6
012345
0 0.2 0.4 0.6 0.8 1Time(hr)CO2( %)
012345
10 15 20 25 30 35 40Time(hr)CO2(%)
Setting Parameters on PC
Culture Cell(Reporter gene transfected )
Addition of Luciferin
ProcedureProcedure
Setting Parameters on PC
Setting Dishes in Kronos
Click Mouse to Start
USB HUBUSB HUB
1 PC Can Control 1 PC Can Control
5 Sets of Kronos5 Sets of Kronos
88 samplessamples 88 samplessamples 88 samplessamples 88 samplessamples 88 samplessamples
4040 samplessamples
Positive elements: Clock, Bmal1
Negative elements: Per1, Per2, Cry1,Cry2
Feedback loop for circadian rhythms
Seoul National Uni. , I. Kwon et al.
Mol. Cell. Biol., 26, 7318-7330 (2006)suprachiasmatic nuclei (SCN)
Per
Cry Repression
Repression
BmalClock
BmalClock
Per mRNA
Cry mRNA
E-boxCry
E-boxCACGTG
Per 1
Dr. S. Honnma, Hokkaido Uni.
Per1 prom. Luciferase
Transfection
2500
Rel
ati
ve
lig
ht
inte
nsi
ty /
45
sec
Monitoring Circadian Rhythm by KronosMonitoring Circadian Rhythm by Kronos
Rat-1
fibroblast cell
Stimulation(by Dex)
Measurement by Kronos
Medium change and
Addition of Luciferin
500
1500
0 1 2 3 4 5 6
DayR
ela
tiv
e li
gh
t in
ten
sity
/ 4
5se
c
DrDrDrDr. Y.Nakajima,National Institute of Advanced Industrial Science and Technology ((((AIST)
Self-sustained circadian rhythms in SCNSCN
SCN section of mouse
transgenic for Per1-Luciferase(300µm)
40000
50000
60000
70000
80000
90000
100000
RL
U/m
in
Dr.Ken-ichi Honma , Hokkaido Uni.
Millicell
Reporter: Per1 Promoter-FLuc
Medium : DMEM, 10mM HEPES, 0.1mM D-Luciferin K
0
10000
20000
30000
0 1 2 3 4 5 6 7 8 9 10 11
Days
RealReal--Time Dual Color Reporter AssayTime Dual Color Reporter Assay
Relative Counts of Per2-Gluc and SV40-RlucRelative Counts of Per2-Gluc
matrix
calculation
Filtered Relative Counts of Per2-Gluc and SV40-Rluc Relative Counts of SV40-Rluc
Dr. Y. Nakajima, AIST
calculation
Cell :NIH3T3
Reporter: Rer2 Promoter-Gluc , SV40-RLuc
Medium : DMEM, 10%FBS
Transfection: Rer2 Promoter-Gluc 1µg , SV40-Rluc 0.2µg
Small Small interference RNAinterference RNA
Control GDF-8-siRNA siRNA(negative contorol)
Fujimori Sato et. al., Am J Physiol Cell Physiol , 291, C538-C545(2006)
Dr. M. Hattori , Kyusyu Uni.
Cell :chicken embryonic myoblasts
Reporter: GDF- 8(Myostatin) Promoter-pGL3 luc
Medium : Opti-MEM, 7.5%knockout serum replacement
Transfection:50pmol GDF-8-siRNA, Lipofectamine2000
Normalization time:20hr
Growth and Differentiation Factor 8(GDF-8): TGF-β superfamily
The promoter activity of growth hormone The promoter activity of growth hormone by Thyroid hormone(T3)by Thyroid hormone(T3)
100nM T3
10nM T3
T. Enomoto, ATTO Corp.
1nM T3
Cell :GH3 (rat pituitary adenoma cell )
Reporter:GH Promoter-Fluc
Medium : OPTI-MEMRef: Y.Tanahashi et.al. Anal. Biochem. 289, 260-266 (2001)
Collective efficiency and transmission and transmission of opitcal systemof opitcal system
Objective Lens
θ
NA η (%)0.1 0.30.15 0.60.2 10.25 1.60.3 2.3
Optics for focusing onto CCD
η =1 − 1 −sin2 θ
2=
1 − 1 −NA2
2
0.3 2.30.35 3.20.4 4.20.45 5.40.5 6.70.55 8.30.6 100.65 120.7 150.75 170.8 201 50NA : numerical aperture (NA=nsinθ)
n : refractive index
η : Collective Efficiency
Cell
High Sensitive & Low Noise Cooled CCDHigh Sensitive & Low Noise Cooled CCD
Exposure time : 10min
Image brightnesson the drawn line
CellgraphCooled CCD Camera
Dark Image
Focus Control
-100 -80 -60 -40
PTGR-Cytosol, (x20 lens)FFocus adjustmentocus adjustment
-20 0 +20 +40
+60 +80 +100
SV40-PTGRm-Cytosol in NIH3T3-3-4 cells
200 mM LH2K in 10% FBS, 25 mM Hepes
X20 lens
3 min exposure20 mmStep
Kronos: 1 x 108 (No.1)
Dr.Y.Nakajima,AIST
Bioluminescence imaging of ELuc and FLuc in NIH3T3 cells
Eluc(New)Fluc(Conventional)
x5.6 lens
The best Luciferase(The best Luciferase(ElucEluc) for cell imaging) for cell imaging
High Quantum Yield in Cell
x5.6 lens
3 min exposure
SV40-PTGRm-Cytosol in NIH3T3-3-4 cells200 mM Luciferin K in 10% FBS, 25 mM Hepes
Medium: DMEM+10%FBS , 200µM D-Luciferin
Dr.Y.Nakajima,AIST
Bioluminescence image of NIH3T3Bioluminescence image of NIH3T3
Transfection of the CMV– Eluc reporter construct into NIH3T3 cells.
×10× 20
CMV promoter – Eluc(2µg)
Transfection : lipofection
Medium: DMEM+10%FBS , 200µM D-Luciferin
Magnification of objective lens :×20, ×10
Exposure time : 3min(××××10)
Dr.Y.Nakajima,AIST
The movie of The movie of Bmal1Bmal1 expressionexpression
175000000
180000000
185000000
190000000
195000000
200000000
205000000
Rela
tiv
e i
nte
nsi
ty
170000000
0 12 24 36 48 60 72
Time (hr)
4hr 16hr 28hr 40hr 52hr 64hrCell : Rat1
Reporter: Bmal1 Promoter-Fluc
Medium : DMEM, 10%FBS, 25mM HEPES,0.2mM D-Luciferin
Magnification of objective lens :×5.2
Exposure time : 20min
Dr. Ken-ichi Honnma ,Hokkaido Uni.
600
800
1000
1200
1400
1600
1800
2000
Rel
ativ
e in
tensi
ty
1
2
3
4
5
6
7
8
9
10
11
Image Analysis Image Analysis
0
200
400
0 24 48 72 96
Time (hr)
11
12
1hr
96 hr
Cell : NIH3T3
Reporter: Bmal1 Promoter-Fluc
Transfection: 2µg
Medium : DMEM, 10%FBS, 25mM HEPES,
0.2mM D-LuciferinMagnification of objective lens :×5.6
Exposure time : 20min
Dr.Y.Nakajima,AIST
mRNA
Transcription
Translation
GenePromorter
DNA Chip
Real-time PCR
Northern Blotting
Reporter Gene Assay
Analysis ToolAnalysis Tool
Translation
Protein
Protein AProtein B
Modification
Complex
Northern Blotting
HPLC
2D-Electrophoresis
MS
Western Blotting
Quantitative Analysis of Quantitative Analysis of mmmmmmmmRNA and ProteinRNA and Protein
Extraction of RNA :PCR , Northern Blotting
Protein: Western Blotting
0 12 24 36 48 52 hr
Monitoring of RealMonitoring of Real--time Changestime Changes
Gen
e E
xp
ress
ion
RealReal--time reporter assaytime reporter assay
Gen
e E
xp
ress
ion
Time
Conventional reporter assayConventional reporter assay
(end(end--point assay)point assay)
Promoter Analysis
Promoter
RLU
Vector
MCS
poly ALuciferasepoly ALuciferin
Promoter
Light
Transfection
50
60
70
80
90
100背面照射型CCD
前面照射型CCDマイクロレンズ付 CCD
量子
効率
(%
)
Quantum Efficiency of CCDQuantum Efficiency of CCD
Back Illuminated CCD
Front Illuminated CCD
QE%
Interline CCD
0
10
20
30
40
200 300 400 500 600 700 800 900 1000
波 長 (nm)
量子
効率
Andor Technology
Wave length (nm)
Endogenous Endogenous
genegene
Analysis of Gene ExpressionAnalysis of Gene Expression
by Luciferase Reporter Assayby Luciferase Reporter Assay
Living CellLiving CellLuciferase geneLuciferase genePromoterPromoter
Reporter vectorReporter vector
PromoterPromoter
mRNAmRNA
LuciferaseLuciferase
Lysis of cellsLysis of cellsMeasurement of Measurement of
bioluminescence bioluminescence
by luminometer by luminometer
Addition of Addition of
luciferin, ATP, Mgluciferin, ATP, Mg2+2+, etc., etc.