Chroma-Luc™ Reporter Vectors - promega.media · prothorax, and a single organ located in the ventral cleft of the abdomen. The dorsal anterior organs emit a yellow to green luminescence,

Revised 7/15TM059

Chroma-Luc™ Reporter VectorsInstruc ons for Use of ProductsE1411, E1421, E1431, E1441, E1451 and E1461

T E C H N I C A L M A N U A L

Chroma-Luc™ Reporter Vectors

All technical literature is available at: www.promega.com/protocols/ Visit the web site to verify that you are using the most current version of this Technical Manual.

E-mail Promega Technical Services if you have questions on use of this system: [email protected]

Promega Corpora on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 1www.promega.com TM059 · Revised 7/15

1. Description .................................................................................................................................. 2

2. Product Components and Storage Conditions ................................................................................. 3

3. General Considerations ................................................................................................................ 3

4. Chroma-Luc™ Reporter Vector Backbones ..................................................................................... 9

5. Emission Color Separation.......................................................................................................... 165.A. Filter Choices .................................................................................................................... 165.B. Data Analysis .................................................................................................................... 16

6. Appendix ................................................................................................................................... 196.A. References ........................................................................................................................ 196.B. Related Products ............................................................................................................... 19

7. Summary of Changes .................................................................................................................. 20

2 Promega Corpora on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516TM059 · Revised 7/15 www.promega.com

1. Description

Normalizing the expression of an experimental reporter to the expression of a control reporter can help to diff erentiate specifi c from nonspecifi c eff ects of cell-treatment protocols. This normalization is helpful for both transiently and stably transfected cell lines. Theoretically, the more similar the experimental and control reporters are, the more relevant the normalization. Based on this principle, we developed a luciferase-based technology, the Chroma-Luc™ Reporter Vectors(a–e). The Chroma-Luc™ Vectors include a vector encoding one red-emitting luciferase (CBRluc) and two vectors encoding green-emitting luciferases (CBG68luc and CBG99luc; Figure 1).

The design of the synthetic Chroma-Luc™ genes is based on a native Yellow-Green luciferase gene originally cloned from Pyrophorus plagiophthalamus, a large click beetle indigenous to the Caribbean. To ensure reliability and high levels of expression, the Chroma-Luc™ genes have been codon optimized for mammalian expression and cleared of most known consensus transcription factor binding sites. In addition, we removed the predicted peroxisome targeting sequences. Upon addition of the luciferase substrates, the engineered CBRluc luciferase emits red light (613 nm), which has optimal color separation from the corresponding 537nm green-emitting Chroma-Luc™ luciferases (i.e., CBG68luc and CBG99luc).

Because of diff erent substrate requirements between the Chroma-Luc™ luciferases and other beetle luciferases, Promega developed Chroma-Glo™ Reagent as a companion reagent to the Chroma-Luc™ Vectors. The Chroma-Glo™ Reagent is designed and optimized to elicit maximal luminescence from all the Chroma-Luc™ enzymes. Color-separating fi lters allow independent measurement of red and green signals.

0

40

60

80

100

120

450 500 550 600 650 700

20

CBG68lucCBG99lucCBRluc

Wavelength (nm)

Perc

ent o

f Max

imum

Lum

ines

cenc

e

4025MA03_3A

Figure 1. Emission spectra of the Chroma-Luc™ enzymes. Independent populations of CHO cells were transfected with either pCBR-Control, pCBG68-Control, or pCBG99-Control. Twenty-four hours after transfection, the cells were lysed by adding Glo Lysis Buff er (Cat.# E2661). Equal volumes of cell lysate and Chroma-Glo™ Reagent were mixed and incubated for 5 minutes at room temperature. The emission spectra were generated using the Spex Fluorolog®-2 spectrofl uorometer with the excitation source turned off . Emission data were collected from 450–700 nm. Maximum emission for CBG68luc and CBG99luc is 537nm while CBRluc has a 537nm, while CBRluc has a maximum emission of 613 nm.


2. Product Components and Storage Conditions

P R O D U C T S I Z E C AT. #

pCBR-Basic Vector 20μg E1411

pCBR-Control Vector 20μg E1421

pCBG68-Basic Vector 20μg E1431

pCBG68-Control Vector 20μg E1441

pCBG99-Basic Vector 20μg E1451

pCBG99-Control Vector 20μg E1461

Storage Conditions: Store the vectors at –20°C.

3. General Considerations

In cell biology research and pharmaceutical discovery, testing a wide variety of experimental conditions or a large number of chemical compounds for their eff ects on cellular physiology is a common practice. This testing is typically achieved through transcriptional assays using genetic reporters coupled to physiologically regulated genetic elements.

Monitoring upregulation of these genetic elements is relatively simple using luciferase assays because they are extremely sensitive, rapid and easy to perform. However, monitoring the downregulation of these genetic elements is more diffi cult because the generalized eff ects of cell death can be interpreted easily as a specifi c decrease in luciferase production. Normalizing the expression of an experimental reporter to the expression of a control reporter can help to distinguish specifi c from nonspecifi c eff ects. This normalization can also reduce the eff ect of variability of the transfection step. The Chroma-Glo™ Luciferase Assay System and the Chroma-Luc™ luciferases provide an ideal dual-reporter system, because the two reporter enzymes are highly similar (>98% amino acid identity) and because both signals are generated by a single-reagent addition.

Pyrophorus plagiophthalamus has two sets of light-emitting organs: a pair located on the dorsal surface of the prothorax, and a single organ located in the ventral cleft of the abdomen. The dorsal anterior organs emit a yellow to green luminescence, while the ventral cleft emits a higher-emission orange luminescence. Four luciferase-expressing genes were cloned from the ventral cleft. The emission for these four luciferases ranges from green to orange (544–593nm). Based on their emission color, the four genes were named Green, Yellow-Green, Yellow and Orange. These four genes express luciferases that are highly similar; the proteins share between 95–99% amino acid identity. Sequence alignment of the four proteins revealed that key amino acids were responsible for the diff erences in emission colors.


3. General Considerations (continued)

The experimental information gathered on how to manipulate emission color was used to design both a synthetic red- and green-emitting luciferase. The wildtype Yellow-Green luciferase demonstrated the brightest signal, and this gene was the template for the Chroma-Luc™ luciferases. In addition to changes to generate diff erent emission colors, several other sequence modifi cations were made to generate the most favorable synthetic genetic reporters To improve the expression, low-usage mammalian codons were replaced in the synthetic Chroma-Luc™ genes.

Table 1. Comparison of Codon Usage Among the Wildtype Yellow-Green Luciferase and the Synthetic CBRluc, CBG99luc and CBG68luc Luciferase Genes.

Amino Acid/Codon

Number of Codons in Yellow-Green Luciferase

Number of Codons in CBRluc (Red)

Number of Codons in CBG99luc (Green)


Percent Use in Human Cells

Ala /GCA GCC

154

412

412

514

20.041.6

GCG GCT

315

120

021

018

10.328.0

Arg/ AGA AGG

77

00

00

00

18.821.0

CGA CGC

61

011

011

211

10.221.4

CGG CGT

04

014

014

013

19.78.9

Asn/ AAC AAT

716

129

139

139

57.742.3

Asp/ GAC GAT

620

1214

1214

1412

57.242.8

Cys/ TGC TGT

49

47

47

38

59.440.6

Gln/ CAA CAG

86

78

77

113

24.875.2

Glu/ GAA GAG

2612

1819

1820

1919

39.360.7

Gly/ GGA GGC

172

321

321

121

24.135.8

GGG GGT

316

214

214

116

24.415.8

His /CAC CAT

67

49

58

76

60.439.6

Ile/ ATA ATC

137

021

020

023

12.954.0

ATT 19 18 18 15 33.1


Amino Acid/Codon

Number of Codons in Yellow-Green Luciferase

Number of Codons in CBRluc (Red)



Percent Use in Human Cells

Leu/ CTA CTC

54

011

011

012

6.520.8

CTG CTT

413

181

181

191

44.411.2

TTA TTG

1813

025

025

023

5.511.5

Lys/ AAA AAG

2512

1222

1322

1916

38.961.1

Met/ ATG 11 10 10 10 100.0

Phe/ TTC TTT

915

1213

1213

1510

58.941.4

Pro/ CCA CCC

98

121

121

92

25.813.0

CCG CCT

29

114

114

017

11.627.3

Ser/ ACG AGT

28

113

132

141

25.735.3

TCA TCC

52

22

22

14

12.824.4

TCG TCT

78

011

012

011

5.818.2

Thr/ ACA ACC

91

211

111

08

25.440.5

ACG ACT

18

011

010

014

11.822.4

Trp/ TGG 2 2 2 2 100.0

Tyr/ TAC TAT

911

137

127

127

60.139.9

Val/ GTA GTC

134

225

125

121

9.325.7

GTG GTT

1119

186

196

263

48.716.4

Total # of Codons 543 542 542 542



To reduce the potential for anomalous expression, virtually all of the known consensus transcription factor binding sites were removed. The wildtype Yellow-Green luciferase gene possessed 152 known consensus tran-scription factor binding sites; the resulting synthetic genes, CBRluc and CBG68luc, have two known consensus transcription factor sites (a 99% reduction), and CBG99luc has four (a 97% reduction; Figure 2). Finally, the synthetic genes express a luciferase enzyme without a peroxisome targeting sequence, and all synthetic genes have a Kozak consensus sequence.

The data in Figure 3 demonstrate that all the synthetic genes exhibit a signifi cant increase in expression over the native gene. Figure 4 demonstrates that the synthetic Chroma-Luc™ genes have reduced anomalous expression.

The data in Figures 3 and 4 are not corrected for photomultiplier (PMT) effi ciency. To obtain a signifi cant amount of the red-emitted light from the expression of CBRluc, the luminometer (more specifi cally, the PMT) must be selected with consideration. Generally a PMT is more sensitive in the blue to green wavelengths and least sensitive in the red wavelengths. A charged coupled device (CCD) instrument works well for detecting the Chroma-Luc™ luciferases because it possesses a detection range that is more sensitive in the red spectrum.

Apart from the diff erent colors of luminescence, the signifi cant diff erences among the three Chroma-Luc™ genes are at the level of DNA. CBG99luc and CBRluc have 99% DNA identity, while CBG68luc and CBRluc have 68.9% DNA identity. The two green-emitting luciferases are expressed from CBG68luc and CBG99luc and are 69.2% identical. The Chroma-Luc™ genes CBRluc and CBG68luc are most dissimilar genetically while retaining a high degree of protein homology. These two genes express proteins that are 98.3% identical. The CBRluc and CBG99luc genes possess a high degree of protein similarity, approximately 98.5%. Finally the proteins expressed by CBG68luc and CBG99luc genes are 99.6% homologous; there is one amino acid diff erence.


LF-A

1

CA

C-b

indi

ng p

rote

inPi

t-la

LVc

NF-

1PE

A3

TFIID

Sp1

AP-3

HNF-3

AR

F2F

YY1IU

F-1

HN

F-1

HiNF-A

E47

Pit-l

a N-O

ct-3

TBP

c-M

tb CD

PA

LF1B

H4T

F-2

TBP

NF-

3

TBP

N-Oct-3

N-Oct

-3

H4TF-2PTF1-betaCDP

NF-EIRF-1Pit-la

NF-ENF-1/L

HNF-3

HNF-3

GATA-1

GATA-1

Tsi-1

Pit-la

C/EBP alpha

GR alpha

GR

H4TF-2 c-Mybc-ETS-2

E12

NF-1

PTF1-beta

E47

Sp1c-ETS-2

c-ETS-2

Myc-CF1

NF-E

Myc-CF1

c-Myb

TBP

NF-1/L

N-Oct-3HiNF-ANF-1

GATA-1GATA-3

GATA-1

Pit-la

Pit-laGR

GR

GR c-Myb

c-Myc

TBPRad-1

LVc

CAC-binding protein

Pit-la

Pit-la

GR

LEF-1AP-3

NF-GMb

E12

Yi Sp1 PPARER

NF-1/LN-Oct-3

N-Oct-3

F2FC/EBP alpha

H4TF-2

AP-3IUF-1

HNRF

GATA-2

ARPR

NF-3

NF-1/L

C/EBP betaC/E

BP beta

C/EBP alpha

GR alp

ha

C/EBP a

lpha

Oct-R

HNF-3

AP-2

Sp1

GR

GR

YYI

TBP

TBP

CAC-binding protein

TFIID

CTF-2

CDPMEF

-2

C/EBP

alph

a

C/EBP

alph

aC/E

BP-b

eta

HiN

F-A

TBP

NF-

1AP

-3Sp

1 Rad-1

E12

gam

ma

CAC

1

Myc

-CF1

LVc

NF-E

CP1GR

GR

YYI

C/EBP al

pha

C-ETS

-2

C/EBP al

pha

F2FGATA

-1

GATA-1

CREB

IUF-1C/E

BP alpha

Pit-la

TBP

Wildtype Yellow-Green luciferase

3965

MA

01_3

A

CBRluc

HES-1

TBP

3966

MA

01_3

A

CBG99luc

HES-1

AP-2

LF-A

1

IUF-1

39

67

MA

01

_3A

CBG68luc HES

-1

HES-1

3968MA01_3A

Figure 2. Known transcription factor binding sites in the wildtype Yellow-Green luciferase gene (top left), red luciferase (CBRluc; top right), green luciferase (CBG99luc; bottom left), and green luciferase (CBG68luc; bottom right). Most (>97%) of the known transcription factor binding sites from the wildtype Yellow-Green gene were removed to create the synthetic Chroma-Luc™ genes. The wildtype Yellow-Green luciferase gene has 152 known transcription factor binding sites.



4056

MA

03_3

A

Wild

type Y

ellow

-Gre

en

Contro

l

pCBG68-C

ontro

l

pCBG99-C

ontro

l

pCBR-Con

trol

10,000

100,000

1,000,000

10,000,000

100,000,000

Lum

ines

cenc

e (R

LU)

Figure 3. Increase in expression from the synthetic Chroma-Luc™ genes. The three synthetic Chroma-Luc™ genes and the wildtype Yellow-Green luciferase gene were independently cloned into the pGL3-Control Vector, replacing the luc+ present in this vector. At twenty-four hours post-transfection of CHO cells an equal amount of Chroma-Glo™ Reagent was added to the medium and the cells. The relative light units were detected using a Berthold Centro LB 960 Luminometer. When compared to the wildtype Yellow-Green luciferases, the synthetic Chroma-Luc™ luciferases, CBG68 and CBG99, display 546- and 660-fold increases in relative light units, respectively. The synthetic CBR luciferase displays a 28-fold increase in relative light units compared to the wildtype Yellow-Green luciferase. All values have been corrected for transfection effi ciency but not for photomultiplier effi ciency.


40

38

MA

03

_3

A

Wild

type Y

-G-B

asic

pCBG68-B

asic

pCBG99-B

asic

pCBR-Bas

ic0

5

10

15

20

25

30

35

40

45

Perc

ent o

f Con

trol V

ecto

rLu

min

esce

nce

39M

A03

_3A

Perc

ent o

f Con

trol V

ecto

rLu

min

esce

nce

Wild

type Y

-G-E

nhan

cer

pCBG68-E

nhan

cer

pCBG99-E

nhan

cer

pCBR-Enh

ance

r0

50

100

150

200

250

300

Figure 4. Reduction in anomalous expression for the synthetic Chroma-Luc™ genes. The synthetic CBRluc, CBG68luc, and CBG99luc genes and the wildtype Yellow-Green luciferase gene were cloned into a pGL3-Control Vector (contains SV40 promoter and enhancer), a pGL3-Enhancer Vector (contains only SV40 enhancer) and pGL3-Basic Vector (no promoter or enhancer added). The vectors were transfected separately into CHO cells. At 24 hours post-transfection, an equal amount of Chroma-Glo™ Reagent was added to the medium and cells. The relative light units were collected using a Berthold Centro LB 960. Expression levels are shown as percentages of the corresponding Control vectors. The data show greatly reduced relative expression of the synthetic reporter in the absence of promoter. Panel A. The luciferase expression from the pGL3-Enhancer Vector with the synthetic Chroma-Luc™ genes was reduced to an average of 1.8% of control. Panel B. The basal level of luciferase transcription from the pGL3-Basic Vector with the synthetic Chroma-Luc™ genes was reduced to an average of 0.6% of control. These experiments were repeated with similar results.

4. Chroma-Luc™ Reporter Vector Backbones

The Chroma-Luc™ Basic Vector series includes pCBR-Basic (Cat.# E1411), pCBG68-Basic (Cat.# E1431) and pCBG99-Basic (Cat.# E1451). No promoter or enhancer sequence has been added to the Basic series. The vector backbone is from the pGL3 series and contains a multiple cloning region for cloning regulator elements of interest.

The Chroma-Luc™ Control Vector series includes pCBR-Control (Cat.# E1421), pCBG68-Control (Cat.# E1441) and pCBG99-Control (Cat.# E1461). These vectors contain an SV40 promoter and SV40 enhancer and have the same vector backbone as the Basic series.

SV40 Promoter and Enhancer

The Control Vectors possess an SV40 Early Promoter and an Enhancer, which have been shown to provide strong, constitutive expression in a variety of cell types. The Basic Vectors do not possess these elements. The Control Vectors contain the SV40 origin of replication within the SV40 promoter. The SV40 origin of replication results in transient, episomal replication in cells expressing the SV40 large T antigen such as COS-1 and COS-7 cells (1).

A. B.


4. Chroma-Luc™ Reporter Vector Backbones (continued)

SV40 poly(A)

Polyadenylation signals cause the termination of transcription by RNA polymerase II and signal the addition of approximately 200–250 adenosine residues to the 3´ end of the RNA transcription (2). Polyadenylation has been shown to enhance RNA stability and translation effi ciency (3,4).

f1 Origin of Replication

To provide the ssDNA template (i.e., for use in mutagenesis) the Chroma-Luc™ Vectors contain an origin of replication derived from fi lamentous phage. This allows single-stranded plasmid DNA to be produced and secreted in phage-like particles from E. coli infected with the appropriate helper phage. Note: Only the sequence of the f1 origin has been verifi ed; no quality control assays were performed to test the functionality of this region.

XbaI 1720

Ampr

KpnISacIMluINheISmaIXhoIBgIIIHindIII

pCBR-BasicVector

(4796 bp)

f1 ori

ori

SalIBamHI

NcoI 86

SV40 latepoly(A) signal

HpaI 1880

511152128323653

19881982

CBRluc3

95

1M

A0

1_

3A

Synthetic poly(A)signal/transcriptional pause site(for background reduction)

Figure 5.The pCBR-Basic Vector circle map. This vector contains: CBRluc, synthetic DNA sequence encoding the red luciferase enzyme; Ampr, gene conferring ampicillin resistance in E. coli; ori, origin of plasmid replication in E. coli. Arrows within the CBRluc and Ampr genes indicate the direction of functionality. Vector sequence is available in the GenBank® database (GenBank®/EMBL Accession Number AY258591) and online at www.promega.com/vectors/

pCBR-Basic Vector Sequence Reference Points:

Multiple cloning region 1–58 CBRluc reporter gene 88–1716

SV40 late poly(A) region 1750–1971 Reporter Vector Primer 4 (RV Primer 4) 2039–2058

ß-lactamase (Ampr) coding region 3058–3918 f1 origin of replication 4050–4505

Synthetic poly(A) signal 4636–4789 Reporter Vector Primer 3 (RV Primer 3) 4738–4757


GCATTCCGGTACTGTTGGTAAAGCCACCATGG

GGTACCGAGCTCTTACGCGTGCTAGCCCGGGCTCGAGATCTGCGATCTAAGTAAGCTTG

KpnIAcc65I

SacI MluI

NcoI

NheI SmaI XhoI BglII HindIII

CBRluc Coding RegionStart

3241

MB

03_1

A

Figure 6. Multiple cloning region of the pCBR-Basic Vector.

XbaI 1912

Ampr

KpnISacIMluINheISmaIXhoIBgIII

pCBR-ControlVector

(5234 bp)

f1 ori

ori

SalIBamHI

NcoI 278

SV40 late poly(A) signal

HpaI 2072

5111521283236

24262420

CBRluc

HindIII 245

SV40 Promoter

SV40 Enhancer3

95

2M

A0

1_

3A


Figure 7. The pCBR-Control Vector circle map. This vector contains: CBRluc, synthetic cDNA sequence encoding the red luciferase enzyme; Ampr, gene conferring ampicillin resistance in E. coli; ori, origin of plasmid replication in E. coli. Arrows within the CBRluc and Ampr genes indicate the direction of functionality. Vector sequence is available in the GenBank® database (GenBank®/EMBL Accession Number AY258592) and online at www.promega.com/vectors/

pCBR-Control Vector Sequence Reference Points:

SV40 promoter 48–250 CBRluc reporter gene 280–1908

SV40 late poly(A) region 1942–2163 SV40 Enhancer 2183–2419

Reporter Vector Primer 4 (RV Primer 4) 2477–2496 ß-lactamase (Ampr) coding region 3496–4356

f1 origin of replication 4488–4943 Synthetic poly(A) signal 5074–5227

Reporter Vector Primer 3 (RV Primer 3) 5176–5195



F

GGTACCGAGCTCTTACGCGTGCTAGCCCGGGCTCGAGATCTGCGATCTGCA

KpnIAcc65I

SacI MluI NheI SmaI XhoI BglIISV40 Promoter Start

5591

MA

igure 8. Multiple cloning region of the pCBR-Control Vector.

XbaI 1734

Ampr

KpnISacIMluINheISmaIXhoIBgIIIHindIII

pCBG68-BasicVector

(4810 bp)

f1 ori

ori

SalIBamHI

NcoI 86

CBG68luc


HpaI 1894


511152128323653

20021996

39

56

MA

01

_3

A

Figure 9. The pCBG68-Basic Vector circle map. This vector contains: CBG68luc, synthetic cDNA sequence encoding the green 68 luciferase enzyme; Ampr, gene conferring ampicillin resistance in E. coli; ori, origin of plasmid replication in E. coli. Arrows within the CBG68luc and Ampr genes indicate the direction of functionality. Vector sequence is available in the GenBank® database (GenBank®/EMBL Accession Number AY258593) and online at www.promega.com/vectors/

pCBG68-Basic Vector Sequence Reference Points:







KpnIAcc65I

SacI MluI

NcoI


CBG68luc Coding RegionStart

3241

MD

03_1

A

Figure10. Multiple cloning region of the pCBG68-Basic Vector.


XbaI 1926

Ampr

KpnISacIMluINheISmaIXhoIBgIII

pCBG68-ControlVector

(5248 bp)

f1 ori

ori

SalIBamHI

CBG68lucSV40 late

poly(A) signalHpaI 2086

Synthetic poly(A) signal/transcriptional pause site (for background reduction)

5111521283236

24402434

NcoI 278

HindIII 245

SV40 Promoter

SV40 Enhancer

39

55

MA

01

_3

A

Figure 11. The pCBG68-Control Vector circle map. This vector contains: CBG68luc, synthetic DNA sequence encoding the green 68 luciferase enzyme; Ampr, gene conferring amplicillin resistance in E. coli; ori, origin of plasmid replication in E. coli. Arrows within the CBG68luc and Ampr genes indicate the direction of functionality. Vector sequence is available in the GenBank® database (GenBank®/EMBL Accession Number AY258594) and online at www.promega.com/vectors

pCBG68-Control Vector Sequence Reference Points:







KpnIAcc65I

SacI MluI NheI SmaI XhoI BglIISV40 Promoter Start

5591

MA

Figure 12. Multiple cloning reagion of the pCBG6-Control Vector.



XbaI 1717

Ampr

KpnISacINheISmaIXhoIBgIIIHindIII

pCBG99-BasicVector

(4793 bp)

f1 ori

ori

SalIBamHI

NcoI 86


HpaI 1877

5112128323653

19851979

CBG99luc

39

53

MA

01

_3

A


Figure 13. The pCBG99-Basic Vector circle map. This vector contains: CBG99luc, synthetic DNA sequence encoding the green 99 luciferase enzyme; Ampr, gene conferring ampicillin resistance in E. coli; ori, origin of plasmid replication in E. coli. Arrows within the CBG99luc and Ampr genes indicate the direction of functionality. Vector sequence is available in the GenBank® database (GenBank®/EMBL Accession Number AY258595) and online at www.promega.com/vectors/

pCBG99-Basic Vector Sequence Reference Points:





F



KpnIAcc65I

SacI MluI*

NcoI


CBG99luc Coding RegionStart

5603

MA

igure 14. Multiple cloning region of the pCBG99-Basic Vector. Note: The MluI site is also present in the CBG99luc gene and should not be used for cloning into the pCBG99-Basic Vector.!


Synthetic poly(A) signal/transcriptional pause site (for background reduction)

XbaI 1909

Ampr

KpnISacINheISmaIXhoIBgIIIpCBG99-Control

Vector(5231 bp)

f1 ori

ori

SalIBamHI

NcoI 278


HpaI 2069

51121283236

24232417

CBG99luc

HindIII 245

SV40 Promoter

SV40 Enhancer

3954

MA

01_3

A

Figure 15. The pCBG99-Control Vector circle map. This vector contains: CBG99luc, synthetic DNA sequence encoding the green 99 luciferase enzyme; Ampr, gene conferring amplicillin resistance in E. coli; ori, origin of plasmid replication in E. coli. Arrows within the CBG99luc and Ampr genes indicate the direction of transcription. Vector sequence is available in the GenBank® database (GenBank® EMBL Accession Number AY258596) and online at www.promega.com/vectors/

pCBG68-Control Vector Sequence Reference Points:







KpnIAcc65I

SacI MluI* NheI SmaI XhoI BglIISV40 Promoter Start

5604

MA

Figure 16. Multiple cloning region of the pCBG99-Control Vector. Note: The MluI site is also present in the CBG99luc gene and should not be used for cloning into the pCBG99-Control Vector.!


5. Emission Color Separation

In order to measure the diff erent luminescent signals, fi lters must be used to separate the red and green Chroma-Luc™ signals.

5.A. Filter Choices

Promega has used 510/60nm and 610nm long-pass fi lters from Chroma Corporation (Chroma Part# D510/60 and E610LP, respectively) to successfully separate the red and green luminescent signals generated by the Chroma-Luc™ luciferases. These fi lters permit transmittance in the spectral region shown in Figure 17.

The 510/60nm and 610nm long-pass fi lter pair provide a balance between sensitivity and separation. This fi lter pair was chosen so that the two colored signals have the least amount of overlap, while permitting transmittance of the largest possible portion of each luminescent spectrum. Other fi lter pairs can be used if extreme separation or extreme sensitivity is critical. If extreme separation of signal is required, fi lter pairs may be chosen so that no green spectrum tailing is captured in the red signal. A 640nm long-pass fi lter, for example, might be chosen for this application and combined with a 510/60nm fi lter.

If extreme sensitivity is critical, fi lter pairs may be chosen so that more of each signal spectrum is transmitted. This means that more of the red luminescent signal will be transmitted through the red fi lter. Regardless of the fi lter set chosen, the luminescent signal transmittance through each fi lter must be calculated. Section 6.B describes the mathematical formulae that are required to separate the red and the green luminescent signals. These calculations must be performed regardless of fi lter choice and must be performed for each combination of fi lter pair and luminometer.

5.B. Data Analysis

Color Separation

Filter sets provide the ability to measure a consistent portion of the red or green luminescence generated by a sample. If the same fi lters are used for all measurements in an experiment, then it is not necessary to calculate the total luminescence generated by each luciferase. The relative proportion that is measured therefore can be used to calculate induction ratios and other experimentally relevant information.

Figure 17 shows the overlap of the spectra generated by the Chroma-Luc™ luciferase enzymes in the transmit-tance window of two fi lters. Filter corrections can be made to factor out the tail of the green luminescent signal from a red luminescent measurement. A single calculation of the fi lter corrections is adequate for all measure-ments using a single luminometer or CCD camera with a particular fi lter set. However, measurements using a diff erent instrument or fi lter set require calculation of diff erent correction factors.


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60

80

100

120

450 500 550 600 650 700

20

CBG68lucCBG99lucCBRluc

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Figure 17. Emission spectra for Chroma-Luc™ luciferases. Separate populations of CHO cells were transiently transfected with the Chroma-Luc™ Vectors. CBRluc is the DNA coding for the red-emitting luciferase, and CBG68luc and CBG99luc are the two diff erent DNA sequences coding for the green-emitting luciferase. At 24 hours post-transfection the cells were removed by trypsin treatment, lysed by the addition of Glo Lysis Buff er (Cat.# E2661) and an equal amount of Chroma-Glo™ Reagent. The spectra data were captured using a Spex Fluorolog®-2 spectrofl uorometer with the excitation source off . Emission data were collected from 450–700nm. Maximum emission for CBG68luc and CBG99luc is 537nm, while CBRluc has a maximum emission of 613nm. The light block indicates the maximal transmittance range of a 610 nm long-pass (greater than 610 nm) fi lter. The dark block indicates the maximal transmittance range of a 510/60 (510±30nm) fi lter.

Calculating the fi lter corrections (calibration constants) requires a one-time measurement of two samples of cells, one expressing only a red-emitting luciferase and the other expressing only a green-emitting luciferase. A total of 6 one-time measurements (Table 2) must be made to determine calibration constants used to normalize fi lter and detector effi ciencies. The calibration constants should not change over time; however, these must be recalculated upon substituting fi lter sets, luminometers or detectors. After these calibration constants are known, the corrected red and green luminescence values (R´ and G´) can be determined using two experimental luminescence values (Lrf and Lgf, see Table 3) using the equations provided below.

R´ =

Lrf – [Lgf × (Grf/Ggf)]

(Rrf/R) – (Rgf/R) × (Grf/Ggf)


5.B. Data Analysis (continued)

The corrected amount of red luminescence in a mixed sample, R´, is

calculated by subtracting the portion of the green luminescence measured using the red fi lter [Lgf × (Grf/Ggf)] from the amount of total luminescence measured with the red fi lter (Lrf) that is normalized for fi lter effi ciencies [(Rrf/R) – (Rgf/R) × (Grf/Ggf)].

G´ =

Lgf – [R´ × (Rgf/R)]

(Ggf/G)

The corrected amount of green luminescence from the same sample, G´, is calculated as the amount of total luminescence measured with the green fi lter (Lgf) minus the amount of corrected red luminescence (R´) measured with the green fi lter [R´ × (Rgf/R)] that has been normalized for fi lter effi ciencies (Ggf/G).

Measurements for the calibration constants Rrf/R, Rgf/R, Grf/Ggf and Ggf/G must be made every time fi lter sets and/or detection equipment changes.

To assist you in the determination of corrected luminescence values, Promega has prepared the Chroma-Luc™ Calculator, a PC-based spreadsheet. This tool is available for download at: www.promega.com/techserv/tools/

Table 2. Descriptions of Initial Measurements Required for Calibration Constants Used to Determine Corrected Experimental Luminescence Values from Overlapping Chroma-Luc™ Spectra.

Name Symbol Measurement

Total Red Luminescence, no fi lter R No fi lter used. Total luminescence measured from the red-emitting luciferase.

Total Green Luminescence, no fi lter G No fi lter used. Total luminescence measured from the green-emitting luciferase.

Red Luminescence, using red fi lter Rrf Luminescence measured from the red-emitting luciferase. Red fi lter used.

Green Luminescence, using red fi lter Grf Luminescence measured from the green-emit-ting luciferase. Red fi lter used.

Red Luminescence, using green fi lter Rgf Luminescence measured from the red-emitting luciferase. Green fi lter used.

Green Luminescence, using green fi lter Ggf Luminescence measured from the green-emit-ting luciferase. Green fi lter used.


Table 3. Experimental Luminescence Values Required to Determine Corrected Luminescence Values, R´ and G´.

Name Symbol Measurement

Experimental luminescence, red fi lter Lrf Experimental luminescence measured with the red fi lter.

Experimental luminescence, green fi lter Lgf Experimental luminescence measured with the green fi lter.

6. Appendix

6.A. References

1. Buchman, A.R. and Berg, P. (1988) Comparison of intron-dependent and intron-independent gene expres-sion. Mol. Cell. Biol. 8, 4395–405.

2. Loeffl er, J.P. et al. (1990) Lipopolyamine-mediated transfection allows gene expression studies in primary neuronal cells. J. Neurochem. 54, 1812–15.

3. Graham, F.L. and van der Eb, A.J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–67.

4. Wigler, M. et al. (1977) Transfer of purifi ed herpes virus thymidine kinase gene to cultured mouse cells. Cell 11, 223–32.

6.B. Related Products

Luciferase Assay System

Product Size Cat.#Chroma-Glo™ Luciferase Assay System 10ml E4910

100ml E4920

10 × 100ml E4950

Lysis Buff er

Product Size Cat.#Glo Lysis Buff er, 1X 100ml E2661

Plasmid DNA Purifi cation System

Product Size Cat.#PureYield™ Plasmid Midiprep System 25 preps A2492

100 preps A2495


© 2003–2015 Promega Corpora on. All Rights Reserved.

Chroma-Glo, Chroma-Luc and PureYield are trademarks of Promega Corpora on.

Fluorolog is a registered trademark of HORIBA Jobin Yvon, Inc. GenBank is a registered trademark of the U.S. Dept. of Health and Human Services

Products may be covered by pending or issued patents or may have certain limita ons. Please visit our Web site for more informa on.

All prices and specifi ca ons are subject to change without prior no ce.

Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date informa on on Promega products.(a) U.S. Pat. Nos. 6,387,675 and 6,552,179, European Pat. No. 0689587 and other patents pending.(b) Certain applica ons of this product may require licenses from others.(c) BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If the researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide the researcher with a full refund.

Researchers may use this product for research use only, no commercial use is allowed. “Commercial use” means any and all uses of this product and deriva ves by a party for money or other considera on and may include but is not limited to use in: (1) product manufacture; and (2) to provide a service, informa on or data; and/or resale of the product or its deriva ves, whether or not such product or deriva ves are resold for use in research. Researchers shall have no right to modify or otherwise create varia ons of the nucleo de sequence of the luciferase gene except that researchers may: (1) create fused gene sequences provided that the coding sequence of the resul ng luciferase gene has no more than four deoxynucleo des missing at the aff ected terminus compared to the intact luciferase gene sequence, and (2) insert and remove nucleic acid sequences in splicing research predicated on the inac va on or recons tu on of the luminescence of the encoded luciferase. No other use or transfer of this product or deriva ves is authorized without the prior express wri en consent of Promega. In addi on, researchers must either: (1) use luminescent assay reagents purchased from Promega for all determina ons of luminescence ac vity of this product and its deriva ves; or (2) contact Promega to obtain a license for use of the product and its deriva ves. Researchers may transfer deriva ves to others for research use provided that at the me of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license. With respect to any uses outside this label license, including any diagnos c, therapeu c or prophylac c uses, please contact Promega for supply and licensing informa on. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA. This label license relates to Promega patents and/or patent applica ons on improvements to the luciferase gene.(d) Patent Pending.

(e) U.S. Pat. No. 7,879,540 and European Pat. No. 1341808.

7. Summary of Changes

The 7/15 version of this technical manual was revised to:

1. Remove expired patent and disclaimer statements and discontinued products.

2. Update the manual to new formatting and design.

Documents

Chroma-Luc™ Reporter Vectors - promega.media · prothorax, and a single organ located in the ventral cleft of the abdomen. The dorsal anterior organs emit a yellow to green luminescence,