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

Bioluminescence Reporter GeneBioluminescence Reporter Gene

Photo: Dr. V. VivianiRailroad worm

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

Output Data on PCOutput Data on PC

※※※※Microsoft Excel is not included the product

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)

Real-time Imaging System in Living Cell

ATTO Cellgraph

IIncubation systemncubation system

Culture Dish

Heat Glass CO2

Objective lens

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

Human genome sequenceHuman genome sequence

ATTO Gel Documentation

Analysis System

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

Photon CountingPhoton Counting

e-

Time

PMT

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

cis elements

Element Analysis

Constitutive Promoter

Vector

MCS

poly ALuciferasepoly Apoly A

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.