promoter analysis tools - promega corporation
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Promoter Analysis ToolspGL4 Luciferase Vectors, Dual-Luciferase®
Assays and more…
Harnessing the power
of bioluminescence to
understand cellular physiology
Tap screen to start
Promoter Analysis Tools
Related Products ReferencesOrdering InformationAssay Principle Experimental Firefly Vectors Control Renilla VectorsIntroduction
Traditional Promoter Dissection
8943
MA
Percent Maximum Luc/Ren0 50% 100%
Criticalregion
LuciferaseUnknown region
Deletion mutagenesis can be used to find functional elements responsible for the regulation of gene transcription. The cloned promoter is inserted upstream of the luciferase gene and the resulting construct transfected into cells to determine the function of the sequence.
Commonly called “promoter bashing”
Promoter Analysis Tools
Related Products ReferencesOrdering InformationExperimental Firefly Vectors Control Renilla VectorsIntroduction Assay Principle
• In a Dual-Luciferase® Assay, both the experimental construct with the promoter of interest and a control construct are introduced into the cell
• The Renilla control vector provides a consistent expression of hRluc for normalization of the Firefly luciferase experimental vector
• The hRluc activity will normalize for conditions of poor transfection efficiency and/or cytotoxicity
• Firefly and Renilla luciferase expression is easily assayed with the Dual-Luciferase® or Dual-Glo® Luciferase Assay Systems
• The roles of the firefly and Renilla luciferases can be reversed.
Assay Principle
ControlRenilla luciferaseconstruct
Rluc
tk
ControlR ill
Experimentalfirefly luciferaseconstruct
luc
RE
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MA
Expression levelvaries with treatment
Expression levelvaries little with treament
1. Tr
ansfe
ct 2
. Culture 2–3 days 3. Treat 4. Dual-Luciferase ® Assay
Promoter Analysis Tools
Related Products ReferencesOrdering InformationAssay Principle Control Renilla VectorsIntroduction Experimental Firefly Vectors
Experimental pGL4 Firefly Luciferase VectorsChoose the functionality you need in your vector
Selectable Marker
– None– Hygror
– Neor
– Puror
Luciferase Gene
– Firefly (luc2) • Rapid Response™ (–P, –CP)– Renilla (hRluc) • Rapid Response™ (–P, –CP)
UpstreamElement
– Multiple cloning region– Promoter/ response elements
4897
MA
Ampr
pGL4 Vectors
ori
SV40 latepoly(A) signal
SV40 earlyenhancer/promoter
Syntheticpoly(A)
Poly(A) block(for background
reduction)
4861
MB
0
50
100
150
200
250
Time (hours)
Fold
Indu
ctio
n
luc 2CPluc 2Pluc 2
0 1 2 3 4 5 6 7
pGL4 Vector backbone was redesigned to remove 75% of cryptic transcription factor binding sites in the pGL3 Vector backbone.
RapidResponse™ luc2 genes have shortened half-lives and the shortened half-lives allow closer coupling to triggering event.
luc2 Firefly Luciferase is the best
• Codon optimized for mammalian cells
• Removal of 90% of cryptic transcription factor binding sites found in luc+ of pGL3
pGL4 Technical Manual
online pGL4 Vector Selector
Promoter Analysis Tools
Related Products ReferencesAssay Principle Experimental Firefly Vectors Control Renilla VectorsIntroduction
www.promega.com
Ordering Information
Experimental pGL4 Firefly Luciferase VectorsVector MCS Luciferase Gene Selectable Marker Cat. #
pGL4.10 [luc2] Vector Y luc2 No E6651
pGL4.11 [luc2P] Vector Y luc2P No E6661
pGL4.12 [luc2CP] Vector Y luc2CP No E6671
pGL4.14 [luc2/Hygro] Vector Y luc2 Hygromycin E6691
pGL4.15 [luc2P/Hygro] Vector Y luc2P Hygromycin E6701
pGL4.16 [luc2CP/Hygro] Vector Y luc2CP Hygromycin E6711
pGL4.17 [luc2/Neo] Vector Y luc2 Neomycin E6721
pGL4.18 [luc2P/Neo] Vector Y luc2P Neomycin E6731
pGL4.19 [luc2CP/Neo] Vector Y luc2CP Neomycin E6741
pGL4.20 [luc2/Puro] Vector Y luc2 Puromycin E6751
pGL4.21 [luc2P/Puro] Vector Y luc2P Puromycin E6761
pGL4.22 [luc2CP/Puro] Vector Y luc2CP Puromycin E6771
Constituitively Expressed
pGL4.13 [luc2/SV40] Vector N luc2 No E6681
pGL4.50 [luc2/CMV/Hygro] Vector N luc2 Hygromycin E1310
pGL4.51 [luc2/CMV/Neo] Vector N luc2 Neomycin E1320
Productsmaybecoveredbypendingorisssuedpatentsormayhavecertainlimitations.Pleasevisitwww.promega.comformoreinformation.ThemethodofrecombinantColeopteraluciferasesiscoveredbyU.S.patentNos.5,583,024;5,674,713;and5,700,673.
Renilla Luciferase Control Vectors Ordering Information >
Promoter Analysis Tools
Related Products ReferencesOrdering InformationAssay Principle Experimental Firefly Vectors Control Renilla VectorsIntroduction Control Renilla Vectors
Renilla Luciferase Control VectorsThe best companions for normalizing firefly luciferase reporters
Ampr
ori
SalIBamHI
17051699
hRluc
SV40 early enhancer/promoter
pGL4.73[hRluc/SV40] Vector
(3921bp)
SV40 late poly(A) signal
4728
MA
Synthetic poly(A) signal/transcriptional pause site (for background reduction)
hRluc Improvements• Codon optimized for mammalian cells
• Removal of 95% of cryptic transcription factor binding sites found in Rluc of pRL
pGL4 Technical Manual
online pGL4 Vector Selector
Ampr
ori
SalIBamHI
20212015
hRluc
HSV-TK promoter
pGL4.74[hRluc/TK] Vector
(4237bp)
SV40 late poly(A) signal
4729
MA
Synthetic poly(A) signal/transcriptional pause site (for background reduction)
Ampr
ori
SalIBamHI
2065 2059
hRluc
CMV immediate- earlyenhancer/ promoter
pGL4.75[hRluc/CMV] Vector
(4281bp)
SV40 late poly(A) signal
4730
MA
Synthetic poly(A) signal/transcriptional pause site (for background reduction)
Promoter Analysis Tools
Related Products ReferencesAssay Principle Experimental Firefly Vectors Control Renilla VectorsIntroduction
www.promega.com
Ordering Information
Renilla Luciferase Control VectorsVector MCS Luciferase
GeneSelectable
Marker Cat. #
pGL4.73 [hRluc/SV40] Vector N hRluc No E6911
pGL4.74 [hRluc/TK] Vector N hRluc No E6921
pGL4.75 [hRluc/CMV] Vector N hRluc No E6931
Promoterless vectors with RapidResponse™ Gene options
pGL4.70 [hRluc] Vector Y hRluc No E6881
pGL4.71 [hRlucP] Vector Y hRlucP No E6891
pGL4.72 [hRlucCP] Vector Y hRlucCP No E6901
Productsmaybecoveredbypendingorisssuedpatentsormayhavecertainlimitations.Pleasevisitwww.promega.comformoreinformation.ThemethodofrecombinantColeopteraluciferasesiscoveredbyU.S.patentNos.5,583,024;5,674,713;and5,700,673.
Ampr
pGL4.70[hRluc] Vector
(3522bp)
ori
13061300
SalIBamHI
hRluc
SV40 late poly(A) signal
Synthetic poly(A) signal/ transcriptional pause site (for background reduction)
4725
MA
SfiIAcc65IKpnIEcoICRISacINheIXhoIBglIISfiIHindIII
9 15 19 24 26 28 34 47 60 66
Experimental pGL4 Firefly Luciferase Vectors
Ordering Information >
Promoter Analysis Tools
ReferencesOrdering InformationAssay Principle Experimental Firefly Vectors Control Renilla VectorsIntroduction Related Products
Product Size Cat. #
Dual Reporter Assays (larger sizes available)
Dual-Luciferase® Reporter Assay System 5 Step assay requiring lysate production. Use in multi-well plates requires dual-injectors
100 assays
E1910
Dual-Glo® Luciferase Assay System 2 step assay that lyses cells directly. Use in multiwell plates does not require injectors.
10ml E2920
Rapid, Transfection-Grade Plasmid Preps
PureYield™ Plasmid Miniprep System 100 preps A1223
PureYield™ Plasmid Midiprep System 25 preps A2492
PureYield™ Plasmid Maxiprep System 10 preps A2392
Transfection Reagent
FuGENE® HD Transfection Reagent*1ml E2311
5x1ml E2312
Dual-Glo,Dual-LuciferaseandGloMaxareregisteredtrademarks;GloResponse,RapidResponseandPureYieldaretrademarksofPromegaCorporation.HighWirePressisaregisteredtrademarkoftheBoardofTrusteesoftheLelandStanfordJuniorUniversity* FuGENE HD is sold only for research use at non-profit entities. See terms of use at www.promega.com/lull
Related Products
GloMax® Multi+ Detection System
Need a luminometer? Go to www.promega.com/glomaxto learn more and request a demo
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TA
Introduction to Reporter Gene Assays Animation
Online pGL4 Vector Selector
Promoter Analysis Tools
Related Products ReferencesOrdering InformationAssay Principle Experimental Firefly Vectors Control Renilla VectorsIntroduction
www.promega.com
References
pGL4.10-pGL4.12 HighWire Press®
pGL4.14-pGL4.22 HighWire Press®
pGL4.70-pGL4.72 HighWire Press®
pGL4.73-pGL4.75 HighWire Press®
References
Citations
Kopish, K. (2007) Deciphering the pGL4 vector code. Promega Notes 96, 6-7.
Paguio, A., et al. (2005) pGL4 Vectors: A new generation of luciferase reporter vectors. Promega Notes 89, 7-10.
Almond, B.D., et al. (2004) Introducing the Rapid Response™ Reporter Vectors. Promega Notes 87, 18-22.
Zhuang, Y., et al. (2001) New synthetic Renilla gene and assay system increase expression, reliability and sensitivity. Promega Notes 79, 6-11.
Schagat, T., Paguio, A. and Kopish, K. (2007) Normalizing genetic reporter assays: Approaches and considerations for increasing consistency and statistical significance. Cell Notes 17, 9-12.
Allard, S.T.M. (2008) Bioluminescent reporter genes. eNotes /fe0030.
Protocols & Applications Guide: Bioluminescent Reporters
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