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1 Real-time Monitoring of Gene Expression in Living Cell by Bioluminescent Detection System
2 Basic Structure of Light detecting equipment Detector Optical system Sample case holder Sample Sample case Light-tight tight box
3 Photon counting Luminometer Photomultiplier tube (PMT) Photoelectric surface Dynode e- Time
4 Quantum efficiency of PMT Quantum efficiency (QE) = electrical signal / incident photon Coelenterazine Luminol Firefly luciferin QE(%) Quantum efficiency 1 Wavelength (nm)
5 Charged Coupled Device (CCD) CCD Pixel Preamp. Readout Horizontal serial register
6 Quantum Efficiency of CCD 率 (%) 量子効率 QE (% %) マイクロレンズ付 Interline CCDC C D Back 背面照射型 Illuminated C C DCCD 前面照射型 Front Illuminated CCD CCD 波長 (nm) Wave length (nm)
7 Basic Structure of Light detecting equipment Detector Optical system Sample case holder Sample ; Living cell Sample case Light-tight tight box
8 Real-time Reporter Assay
9 Promoter Analysis of Gene Expression by Reporter Assay Luciferase gene Living Cell Reporter vector Promoter Endogenous gene Addition of Luciferin, ATP, Mg 2+, etc. Luciferase mrna Luminometer Lysis of cells Bioluminescence
10 Monitoring of Time Course Conventional reporter assay (end-point assay) Gene Exp pression Lysis & Measure Time
11 Monitoring of Time Course Conventional reporter assay Real-time PCR, Northern blot Real-time reporter assay Si ignal Intensity Sig gnal Intensity Sample Time Lysis & Measure Lysis & Measure Lysis & Measure Lysis & Measure Lysis & Measure Lysis & Measure Lysis & Measure Lysis & Measure Time Continuous measuring (without lysis) Sample Lysis & Measure Time Time
12 Real-time Reporter Assay Promoter Luciferase gene Living Cell Reporter vector Luciferin (in the culture medium) Continuous and real-time measurement of light Luciferase mrna Endogenous gene Intensity Time
13 Luminometer for Real-time Reporter Assay Incubator (37, 5% CO2, >95% humidity) in Living Cell 35mm Culture dish 8 Culture Dishes on Turntable Heat insulator Lens & Filters PMT Kronos (AB-2500)
14 Data Display on PC
15 Circadian Rhythm Monitoring Per1 prom. in Culture Cell Luciferase Stimulation Transfection Medium change and Addition of Luciferin Rat-1 fibroblast cell Relative light intensity / 45sec Day Measurement Y. Nakajima, National Institute of Advanced Industrial Science and Technology (AIST), Japan
16 Effect of Thyroid Hormone(T3) to Growth Hormone Promoter Activity Addition of T3 25 Relative activity to 0nM T nM T3 10nM T3 1nM T3 T3 (-) Time (hour) Cell : GH3 (rat pituitary adenoma cell ) Reporter : Growth hormone promoter - Luciferase
17 Multi-color detection
18 Multi-Color Reporter Assay Promoter 1 Red-Luciferase Promoter 2 Orange-Luciferase Promoter 3 Green-Luciferase D-Luciferin Cell Lysis of Cells Red Orange Green Y. Nakajima, et al., FEBS Lett. 565, p (2004) Bio Techniques 38, p (2005)
19 Conventional Multi-color Detection Filter 1 Filter 2 The loss of luminescence by filter is high.
20 New Method of Multi-color Detection No filter Filter 1 The loss of luminescence by filter is very low. Calculations The calculated values were proportional to concentrations of each luciferase.
21 Sample Advanced Method of Quantitative Color Detection F0 (no filter) F1 F2 F G Optical filter unit F1 = κf1gκf1oκf1rf1r O F0 F1 F2 F2 κf2gκf2oκf2rf2r R PMT F : Measurement value κ : Transmittance of each color light in each optical filter G, O, R : Each color light
22 Real-time Multi-color Reporter Assay Filter PMT
23 Real-time Multi Color Reporter Assay without filter Cry1 - Green Luc Bmal1 - Orange Luc Relative light intensity (RLU/min) (Raw Data) matrix calculation Red Luc and Orange Luc (RLU/min) Green Luc (RLU/min) O56 filter Time (hr) R62 filter Cell :NIH3T3 Reporter : Cry1 promoter-eluc (Green luciferase), Bmal1 promoter-slo (Orange luciferase), Per2 promoter-slr (Red luciferase) Per2 - Red Luc Time (hr) Y. Nakajima, AIST, Japan 0
24 Bioluminescent imaging in living cell
25 Mass? or Individual? Luminometer Imaging System Photomultiplier Tube Cooled CCD Intensity Intensity Time Monitoring of Total Cells Time Monitoring of Individual Cell
26 Bioluminescent Imaging System Key Point (1) High sensitivity (2) Blocking of Outside light (3) Long term cell culturing
27 Bioluminescent Imaging System Key Point (1) High sensitivity Detector ( ; CCD camera) S/N Signal ; Quantum efficiency Noise ; Thermal noise, Readout noise, etc. Optical system NA (numerical aperture) Optical transparency
28 Quantum Efficiency of CCD 率 (%) 量子効率 QE (% %) Interline CCD マイクロレンズ付 C C D Back 背面照射型 Illuminated C C DCCD 前面照射型 Front Illuminated CCD CCD 波長 (nm) Wave length (nm)
29 Reduction of Thermal Noise Image brightness on the drawn line Dark Image
30 Readout Noise CCD Pixel Preamp. Readout Horizontal serial register
31 Electron Multiplier CCD Conventional FT-CCD EM-CCD Pixel Pixel Preamp. Readout Horizontal serial register LED Measurement data EM gain OFF Charge multiplier register EM gain x1000 Horizontal serial register Readout LED applied voltage (relative value) x 1 x 1/100 x 1/10,000
32 Light collecting efficiency η= 1 1 sin2 θ 1 1 NA2 = 2 2 η:light collecting efficiency Imaging optics to CCD NA η (%) θ Objective Lens Sample NA:numerical aperture (NA=nsinθ) n : refractive index
33 Optical transparency of optical system Detector Optical system Sample Microscope Objective lens Imaging lens Optical filters Mirrors Dichroic mirrors Correcting lenses Long optical path
34 Bioluminescent Imaging System Key Point (1) High sensitivity (2) Blocking of Outside light (3) Long term cell culturing
35 Blocking of Outside Light Sample stage and Optical system into Dark box
36 Incubation System for Long Term Cell Culturing Glass heater Culture Dish 5% CO 2 Hot air from heater Objective lens
37 Single Cell Bioluminescent Imaging System Cellgraph Luciferase expressed NIH3T3 cells
38 Bioluminescent Imaging Movie Cell : NIH3T3 Reporter: SV40 Promoter-ELuc Transfection: 2µg DNA Medium : DMEM, 10%FBS, 0.2mM D-Luciferin Magnification of objective lens : 20x Exposure time : 20min
39 Bioluminescent Imaging Movie Cell : NIH3T3 Reporter: SV40 Promoter-Eluc Transfection: 2µg DNA Medium : DMEM, 10%FBS, 0.2mM D-Luciferin Magnification of objective lens : 20x Exposure time : 20min
40 Image Analysis 2000 Relative intensity Time (hr) 1hr 96 hr Cell : NIH3T3 Reporter: Clock gene(bmal1) Promoter-Fluc Transfection: 2µg DNA Medium : DMEM, 10%FBS, 25mM HEPES, 0.2mM D-Luciferin Magnification of objective lens : 5.6 Exposure time : 20min Y. Nakajima, AIST, Japan
41 Bioluminescent Imaging Mouse brain SCN tissue culture 1mm Cell : TG mouse brain slice Reporter: Bmal1 Promoter-Eluc Magnification of objective lens : 4x Exposure time : 3min Y. Nakajima (AIST, Japan)
42 Bioluminescent Imaging Luciferase NLS Movie Movie Cell : NIH3T3 Reporter: SV40 Promoter-ELuc-NLS Transfection: 2µg DNA Medium : DMEM, 10%FBS, 0.2mM D-Luciferin Magnification of objective lens : 20x Exposure time : 10min Y. Nakajima, AIST, Japan
43 High intensity bioluminescent probe Firefly luciferase Eluc SV40-ELuc in NIH3T3 cells Medium: DMEM+10%FBS, 0.2mM D-Luciferin V. Viviani, et al., Photochem. Photobiol. 70(2), p (1999)
44 Enhanced emission probe with BRET Movie Bioluminescent Resonance Energy Transfer EYFP Renilla Luc Linker BAF-Y (BRET-based autoilluminated fluorescent protein on EYFP) Cell : NIH3T3 Reporter: CMV Promoter-eBAF-Y Medium : DMEM, 10%FBS, 0.06mM Coelenterazine (ViviRen TM ) Magnification of objective lens : 20x Exposure time : 18sec H. Hoshino, et al., Nature Methods 4(8), p (2007)
45 Summary (1) Real-time reporter assay enable to monitor continuously the time course of gene expression level after drug stimulation in living cells. (2) Our new multicolor bioluminescent detection method enable to monitor the activities of different promoters simultaneously in the same sample. (3) We have developed living cell bioluminescent imaging system. This system is effective to quantitatively trace gene expression of single cell for long term.
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