2. Real-time reporter gene assay by photon counting system. 3. Real-time reporter gene assay by imaging system.

Transcription

1 Real-time Reporter Gene Assay Progress of real-time reporter assay 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.

2 Feature of Bioluminescence Reporter Assay Photo: Dr. Osamu Shimomura Aequorea aequorea Photo: Dr. K. Ogho Valugula hilugendorfii GFP Firefly Photo: Dr. K. Niwa Luciola lateralis

3 Bioluminescence Reporter Gene Photo: Dr. V. Viviani Railroad worm

4 Oxidation of Luciferin & Coelenterazine Luciferin 620nm 560nm Provided by Dr.H.Akiyama Coelenterazine Coelenteramide O 2 Renilla Luciferase +CO 2 +hν

5 Dual Reporter Assay Promoter A Luciferase Promoter B Renilla Luciferase D-Luciferin Coelenterazine Cell Lysis of cells Luciferase Activity Renilla Luciferase Activity

6 Multi-Color Reporter Assay Promoter 1 Red-Luciferase Promoter 2 Orange-Luciferase Promoter 3 Green-Luciferase Cell Culture D-Luciferin measurement of Luminescence Intensity Red Orange Lysis of Cells Green

7 Advanced Method of Quantitative Color Detection Sample Optical filter unit F0 PMT Relative Counts (F0) = G+O+R

8 Advanced Method of Quantitative Color Detection Sample Optical filter unit F1 Relative Counts (F1) = Κf 1g G+Κf 1o O+Κf 1r R PMT Κf1g : Transmittance of green luminescence of f1 filter

9 Advanced Method of Quantitative Color Detection Sample Optical filter unit F0 F2 PMT Relative Counts (F2) = Κf 2g G+Κf 2o O+Κf 2r R

10 Advanced Method of Quantitative Color Detection Sample F G Optical filter unit F1 = κf1gκf1oκf1rf1r O F0 F1 F2 F2 κf2gκf2oκf2rf2r R PMT F : Measurement value κ : Transmittance of each color light of each optical filter G, O, R : Each color light

11 Promoter Real-Time Reporter Assay (Live Cell Reporter Assay) Luciferase gene Living Cell Reporter vector Luciferin (in the culture medium) Continuous and real-time measurement of light Luciferase mrna Endogenous gene Intensity Time

12 Luminometer for real-time reporter assay in living cell Kronos (AB-2500) 35mm Culture Dishes on Turntable Keeping Constant Temperature (20-45 ) Photomultiplier Tube

13 Temperature Stability Sensor in dish 40 Setting on the new dish 35 Dish temp Sensor in kronos 30 Temp( ) Temp( ) Room temp Tim e(sec) /12/26 12:00: /12/28 12:00: /12/ /01/01 12:00:00 12:00:00 Time and Date 2007/01/03 12:00: /01/05 12:00:00 Preset Temperature : 37

14 CO 2 Concentration ) % 3 ( 2 O C ) % ( 2 O C Time(hr) Time(hr)

15 Procedure Addition of Luciferin Culture Cell (Reporter gene transfected ) Setting Parameters on PC Setting Dishes in Kronos Click Mouse to Start

16 Output Data on PC Microsoft Excel is not included the product

17 1 PC Can Control 5 Sets of Kronos USB HUB 8 samples 8 samples 8 samples 8 samples 40 samples 8 samples

18 Feedback loop for circadian rhythms Positive elements: Clock, Bmal1 Negative elements: Per1, Per2, Cry1,Cry2 suprachiasmatic nuclei (SCN) Seoul National Uni., I. Kwon et al. Mol. Cell. Biol., 26, (2006) Per Per mrna Cry mrna Per 1 Clock Repression Bmal E-box CACGTG Cry Cry Clock Bmal E-box Repression Dr. S. Honnma, Hokkaido Uni.

19 Monitoring Circadian Rhythm by Kronos Per1 prom. Luciferase Transfection Stimulation(by Dex) Medium change and Addition of Luciferin Rat-1 fibroblast cell Relative light intensity / 45sec Day Measurement by Kronos Dr. Y.Nakajima, National Institute of Advanced Industrial Science and Technology (AIST)

20 Self-sustained circadian rhythms in SCN SCN section of mouse transgenic for Per1-Luciferase (300µm) RLU/min Millicell Days Dr.Ken-ichi Honma, Hokkaido Uni. Reporter: Per1 Promoter-FLuc Medium : DMEM, 10mM HEPES, 0.1mM D-Luciferin K

21 Real-Time Dual Color Reporter Assay Relative Counts of Per2-Gluc and SV40-Rluc Relative Counts of Per2-Gluc matrix calculation Filtered Relative Counts of Per2-Gluc and SV40-Rluc Cell :NIH3T3 Reporter: Rer2 Promoter-Gluc, SV40-RLuc Medium : DMEM, 10%FBS Transfection: Rer2 Promoter-Gluc 1µg, SV40-Rluc 0.2µg Relative Counts of SV40-Rluc Dr. Y. Nakajima, AIST

22 Small interference RNA Control GDF-8-siRNA sirna(negative contorol) Growth and Differentiation Factor 8(GDF-8): TGF-β superfamily 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 Fujimori Sato et. al., Am J Physiol Cell Physiol, 291, C538-C545(2006) Dr. M. Hattori, Kyusyu Uni.

23 The promoter activity of growth hormone by Thyroid hormone(t3) 100nM T3 10nM T3 1nM T3 Cell :GH3 (rat pituitary adenoma cell ) Reporter:GH Promoter-Fluc Medium : OPTI-MEM T. Enomoto, ATTO Corp. Ref: Y.Tanahashi et.al. Anal. Biochem. 289, (2001)

24 Real-time Imaging System in Living Cell ATTO Cellgraph

25 Incubation system Culture Dish Heat Glass CO 2 Objective lens

26 Collective efficiency and transmission of opitcal system Optics for focusing onto CCD η = 1 1 sin2 θ 2 η : Objective Lens θ = 1 1 NA2 2 Collective Efficiency Cell NA : numerical aperture (NA=nsinθ) n : refractive index NA η (%)

27 High Sensitive & Low Noise Cooled CCD Exposure time : 10min Image brightness on the drawn line Dark Image Cooled CCD Camera Cellgraph

28 Focus adjustment PTGR-Cytosol, (x20 lens) Focus Controｌ Dr.Y.Nakajima AIST SV40-PTGRm-Cytosol in NIH3T3-3-4 cells 200 mm LH2K in 10% FBS, 25 mm Hepes +100 X20 lens 3 min exposure 20 mmstep Kronos: 1 x 108 (No.1)

29 The best Luciferase(Eluc) for cell imaging Bioluminescence imaging of ELuc and FLuc in NIH3T3 cells Fluc(Conventional) Eluc(New) x5.6 lens 3 min exposure High Quantum Yield in Cell Dr.Y.Nakajima,AIST SV40-PTGRm-Cytosol in NIH3T3-3-4 cells 200 mm Luciferin K in 10% FBS, 25 mm Hepes Medium: DMEM+10%FBS, 200µM D-Luciferin

30 Bioluminescence image of NIH3T3 Transfection of the CMV Eluc reporter construct into NIH3T3 cells 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

31 The movie of Bmal1 expression Relative intensity Time (hr) Dr. Ken-ichi Honnma,Hokkaido Uni. 4hr 16hr 28hr 40hr 52hr 64hr Cell : Rat1 Reporter: Bmal1 Promoter-Fluc Medium : DMEM, 10%FBS, 25mM HEPES, 0.2mM D-Luciferin Magnification of objective lens : 5.2 Exposure time : 20min

32 Image Analysis 2000 Relative intensity Time (hr) 1hr Dr.Y.Nakajima,AIST 96 hr Cell : NIH3T3 Reporter: Bmal1 Promoter-Fluc Transfection: 2µg Medium : DMEM, 10%FBS, 25mM HEPES, 0.2mM D-Luciferin Magnification of objective lens : 5.6 Exposure time : 20min

33 Human genome sequence ATTO Gel Documentation Analysis System

34 Analysis Tool Promorter Gene Transcription Reporter Gene Assay Translation mrna DNA Chip Real-time PCR Northern Blotting Protein Modification Complex Protein A Protein B HPLC 2D-Electrophoresis MS Western Blotting

35 Photon Counting e- Time PMT

36 Quantitative Analysis of mrna and Protein Extraction of RNA :PCR, Northern Blotting Protein: Western Blotting hr

37 Monitoring of Real-time Changes Gen ne Expression Real-time reporter assay Time Conventional reporter assay (end-point assay)

38 Promoter Analysis RLU Promoter poly A Luciferase poly A Light Luciferin MCS Vector Transfection

39 Element Analysis cis elements Constitutive Promoter poly A MCS Luciferase poly A Vector

40 Quantum Efficiency of CCD 量子子効率 (%) QE% Interline CCD マイクロレンズ付 C C D Back Illuminated CCD 背面照射型 C C D Front 前面照射型 Illuminated CCD 波長 (nm) Wave length (nm) Andor Technology

41 Analysis of Gene Expression by Luciferase Reporter Assay Promoter Luciferase gene Living Cell Reporter vector Promoter Endogenous gene Addition of luciferin, ATP, Mg 2+, etc. Luciferase mrna Measurement of bioluminescence by luminometer Lysis of cells