荧光素酶报告系统 luciferase reporter assay design
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• • • •
Sequence optimized
codon utilization & “cleaned”
Sequence optimized
codon utilization & “cleaned”
~200-fold increased signal compared to native luc per gene copy number Luc2 protein half-life ~ 3 hr Further destabilized version, Luc2P, ~1.5 hr protein half-life
Firefly luciferase - Luc2
61 kDa, ATP-dependent enzyme; utilizes beetle luciferin (D-luciferin)
• • • •
NanoLuc® - Nluc
19 kDa, ATP-independent enzyme; utilizes novel coelenterazine analog (furimazine)
•
• •
No endogenous analog (protein or substrate) – low background
Convenient assay Quantitative, sensitive & wide dynamic range
CAT β-gal GFP luciferase
Promega Corporation
Fold Expression
Protein
fusion
post-translational
REPORTER ASSAY
Figure 1
REPORTER CONSTRUCT
Promega Corporation
©2014 Promega Corporation.
8
Reporters Can Be Used For A Variety Of Applications
(the substrate is generically referred to as luciferin)
luciferin + O2
luciferase
co-factor
Oxidized luciferin +
LIGHT
Light signal is read by a luminometer or a CCD camera
Fold Expression
3’-UTR
fusion
post-transcriptional
REPORTER ASSAY
Figure 1
translation REPORTER CONSTRUCT
Promega Corporation
©2014 Promega Corporation.
7
Translational Fusion To The Reporter Gene
Response Element fusion
transcription
REPORTER ASSAY
Figure 1
REPORTER CONSTRUCT
Promega Corporation
©2014 Promega Corporation.
6
Transcriptional Fusion To The Reporter Gene
Reporter as a translational fusion
•
• •
Genetic Reporter.
Reporter as a transcriptional fusion
•
Short protein half-life
maximally responsive
Small, compact protein
©2014 Promega Corporation.
11
Bioluminescence In Nature
Marine algae (Gonyaulax)
Lantern fish (Lampanyctodes)
Squid (Euprymna) bacteria (Vibrio)
Mushroom (Armillaria)
• Reporters may be used in cells, tissues, or whole
organisms
Promega Corporation
©2014 Promega Corporation.
4
Basic Reporter Assay Principle
Fold Expression
Transgenesis
Cell Culture Detection Assay
Promega Corporation
©2014 Promega Corporation.
Properties Of A Good Reporter
• •
Enzyme - signal amplification Active upon synthesis - no processing or assembly
Lytic
Promega Corporation
©2014 Promega Corporation.
-OR-
Live Assay
13
Properties Of A Good Reporter
• • • • •
Enzyme - signal amplification Active upon synthesis - no processing or assembly No endogenous analog (protein or substrate) – low background Convenient assay Quantitative, sensitive & wide dynamic range Protein Fusion Reporter
Understanding Luminescent Reporter Assay Design
Carl Strayer, PhD
Promega Corporation
©2014 Promega Corporation.
Reporter Assay Design Considerations
Reporter Design
Activity is minimally affected by fusion Protein half-life & localization determined by fusion partner
14
Promega Corporation
©2014 Promega Corporation.
Promega Optimized Luciferase Reporters
10 1 0
NanoLuc™ In cells
is ~100-fold brighter
10 8
10 6
10
4
Fra Baidu bibliotek
Hela / NLuc Hela / FLuc HepG2 / NLuc HepG2 / FLuc
10 2 10 0
10 2
10 4
10 6
Transfected DNA (pg)
Recombinant NLuc/Nano-Glo™ Assay Recombinant FLuc/ONE-Glo™ Assay Recombinant RLuc/Renilla-Glo™ Assay
Promega Corporation
©2014 Promega Corporation.
15
Nanoluc® Provides The Brightest Signal, Best Sensitivity
Purified NanoLuc™
is ~100-fold brighter
Luminescence (RLU)
TREATMENT
REPORTER ASSAY
Figure 1
REPORTER CONSTRUCT
Promega Corporation
©2014 Promega Corporation.
5
Transcriptional Fusion to the Reporter Gene
Promoter
or
Fold Expression
Further ~100-fold increase in signal compared to luc/luc2 per protein copy number Nluc protein half-life >6 hr NlucP destabilized version, ~30 minute protein half-life
©2014 Promega Corporation.
What is a Reporter?
• Genetic reporters are indicators of gene expression
or cellular events coupled to gene expression
• Reporters can mark any gene product
• Transcriptional Fusion – reports on transcriptional and post-transcriptional regulatory inputs & events
• Translational Fusion – reports on post-translational regulatory inputs & events
Promega Corporation
©2014 Promega Corporation.
Glow worms (Arachnocampa)
Jellyfish (Aequorea)
12
Bioluminescence In The Lab - Luciferase As A Reporter
Luciferase is the generic name for the class of enzyme
Gene expression:
Transcription and posttranscriptional regulation
• Promoters/response elements • Enhancers • 5’- and 3’-UTRs • Transcriptions factors • RNA binding proteins & miRNAs
CMV-driven NLuc/Nano-Glo™ Assay CMV-driven FLuc/ONE-Glo™ Assay
Promega Corporation
©2014 Promega Corporation.
9
Promega Corporation
©2014 Promega Corporation.
Reporter Assay Design Considerations
Reporter Choice & Reporter Design
Reporter Design
Reporter Choice Controls
Cellular Events:
Receptor activation/signaling
• Receptor ligands, agonists & antagonists • Nuclear receptors
Pathway analysis
• Defining pathways • Protein:protein interactions
Post-translational regulation
• Protein stability • Protein localization • Protein:protein interactions
Disease/Immune responses
• Cellular response to infection • Cellular response to therapy • Infectious agent replication/response to therapy
Reporter Choice
Controls
Good Data
Transgenesis Cell Culture Detection Assay
Promega Corporation
©2014 Promega Corporation.
2
Reporter Assay Basic Concepts
Promega Corporation
Sequence optimized
codon utilization & “cleaned”
Sequence optimized
codon utilization & “cleaned”
~200-fold increased signal compared to native luc per gene copy number Luc2 protein half-life ~ 3 hr Further destabilized version, Luc2P, ~1.5 hr protein half-life
Firefly luciferase - Luc2
61 kDa, ATP-dependent enzyme; utilizes beetle luciferin (D-luciferin)
• • • •
NanoLuc® - Nluc
19 kDa, ATP-independent enzyme; utilizes novel coelenterazine analog (furimazine)
•
• •
No endogenous analog (protein or substrate) – low background
Convenient assay Quantitative, sensitive & wide dynamic range
CAT β-gal GFP luciferase
Promega Corporation
Fold Expression
Protein
fusion
post-translational
REPORTER ASSAY
Figure 1
REPORTER CONSTRUCT
Promega Corporation
©2014 Promega Corporation.
8
Reporters Can Be Used For A Variety Of Applications
(the substrate is generically referred to as luciferin)
luciferin + O2
luciferase
co-factor
Oxidized luciferin +
LIGHT
Light signal is read by a luminometer or a CCD camera
Fold Expression
3’-UTR
fusion
post-transcriptional
REPORTER ASSAY
Figure 1
translation REPORTER CONSTRUCT
Promega Corporation
©2014 Promega Corporation.
7
Translational Fusion To The Reporter Gene
Response Element fusion
transcription
REPORTER ASSAY
Figure 1
REPORTER CONSTRUCT
Promega Corporation
©2014 Promega Corporation.
6
Transcriptional Fusion To The Reporter Gene
Reporter as a translational fusion
•
• •
Genetic Reporter.
Reporter as a transcriptional fusion
•
Short protein half-life
maximally responsive
Small, compact protein
©2014 Promega Corporation.
11
Bioluminescence In Nature
Marine algae (Gonyaulax)
Lantern fish (Lampanyctodes)
Squid (Euprymna) bacteria (Vibrio)
Mushroom (Armillaria)
• Reporters may be used in cells, tissues, or whole
organisms
Promega Corporation
©2014 Promega Corporation.
4
Basic Reporter Assay Principle
Fold Expression
Transgenesis
Cell Culture Detection Assay
Promega Corporation
©2014 Promega Corporation.
Properties Of A Good Reporter
• •
Enzyme - signal amplification Active upon synthesis - no processing or assembly
Lytic
Promega Corporation
©2014 Promega Corporation.
-OR-
Live Assay
13
Properties Of A Good Reporter
• • • • •
Enzyme - signal amplification Active upon synthesis - no processing or assembly No endogenous analog (protein or substrate) – low background Convenient assay Quantitative, sensitive & wide dynamic range Protein Fusion Reporter
Understanding Luminescent Reporter Assay Design
Carl Strayer, PhD
Promega Corporation
©2014 Promega Corporation.
Reporter Assay Design Considerations
Reporter Design
Activity is minimally affected by fusion Protein half-life & localization determined by fusion partner
14
Promega Corporation
©2014 Promega Corporation.
Promega Optimized Luciferase Reporters
10 1 0
NanoLuc™ In cells
is ~100-fold brighter
10 8
10 6
10
4
Fra Baidu bibliotek
Hela / NLuc Hela / FLuc HepG2 / NLuc HepG2 / FLuc
10 2 10 0
10 2
10 4
10 6
Transfected DNA (pg)
Recombinant NLuc/Nano-Glo™ Assay Recombinant FLuc/ONE-Glo™ Assay Recombinant RLuc/Renilla-Glo™ Assay
Promega Corporation
©2014 Promega Corporation.
15
Nanoluc® Provides The Brightest Signal, Best Sensitivity
Purified NanoLuc™
is ~100-fold brighter
Luminescence (RLU)
TREATMENT
REPORTER ASSAY
Figure 1
REPORTER CONSTRUCT
Promega Corporation
©2014 Promega Corporation.
5
Transcriptional Fusion to the Reporter Gene
Promoter
or
Fold Expression
Further ~100-fold increase in signal compared to luc/luc2 per protein copy number Nluc protein half-life >6 hr NlucP destabilized version, ~30 minute protein half-life
©2014 Promega Corporation.
What is a Reporter?
• Genetic reporters are indicators of gene expression
or cellular events coupled to gene expression
• Reporters can mark any gene product
• Transcriptional Fusion – reports on transcriptional and post-transcriptional regulatory inputs & events
• Translational Fusion – reports on post-translational regulatory inputs & events
Promega Corporation
©2014 Promega Corporation.
Glow worms (Arachnocampa)
Jellyfish (Aequorea)
12
Bioluminescence In The Lab - Luciferase As A Reporter
Luciferase is the generic name for the class of enzyme
Gene expression:
Transcription and posttranscriptional regulation
• Promoters/response elements • Enhancers • 5’- and 3’-UTRs • Transcriptions factors • RNA binding proteins & miRNAs
CMV-driven NLuc/Nano-Glo™ Assay CMV-driven FLuc/ONE-Glo™ Assay
Promega Corporation
©2014 Promega Corporation.
9
Promega Corporation
©2014 Promega Corporation.
Reporter Assay Design Considerations
Reporter Choice & Reporter Design
Reporter Design
Reporter Choice Controls
Cellular Events:
Receptor activation/signaling
• Receptor ligands, agonists & antagonists • Nuclear receptors
Pathway analysis
• Defining pathways • Protein:protein interactions
Post-translational regulation
• Protein stability • Protein localization • Protein:protein interactions
Disease/Immune responses
• Cellular response to infection • Cellular response to therapy • Infectious agent replication/response to therapy
Reporter Choice
Controls
Good Data
Transgenesis Cell Culture Detection Assay
Promega Corporation
©2014 Promega Corporation.
2
Reporter Assay Basic Concepts
Promega Corporation