Protocols and Applications Guide (US Letter Size) - Promega
Protocols and Applications Guide (US Letter Size) - Promega
Protocols and Applications Guide (US Letter Size) - Promega
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|||||||| 8Bioluminescence Reporters<br />
Relative Light Units<br />
10,000,000<br />
1,000,000<br />
100,000<br />
10,000<br />
1,000<br />
Native (Rluc)<br />
Synthetic (hRluc)<br />
CHO HeLa<br />
Cell Line<br />
Figure 8.2. The synthetic Renilla luciferase gene supports higher expression than the native Renilla gene in mammalian cells. CHO<br />
<strong>and</strong> HeLa cells were transfected with pGL3-Control Vector (containing SV40 enhancer/promoter) harboring either the synthetic hRluc or<br />
native Rluc gene. Cells were harvested 24 hours after transfection <strong>and</strong> Renilla luciferase activity assayed using the Dual-Luciferase® Reporter<br />
Assay System.<br />
Luminescence<br />
(Relative Light Units)<br />
10,000,000<br />
1,000,000<br />
100,000<br />
10,000<br />
1,000<br />
7.9<br />
NIH/3T3<br />
4.9<br />
CHO<br />
4.1<br />
luc+<br />
luc2<br />
HEK 293<br />
Figure 8.3. The firefly luc2 gene displays higher expression than the luc+ gene. The luc2 gene was cloned into the pGL3-Control Vector<br />
(Cat.# E1741), replacing the luc+ gene. Thus both firefly luciferase genes were in the same pGL3-Control Vector backbone. The two vectors<br />
containing either of the firefly luciferase genes were co-transfected into NIH/3T3, CHO, HEK 293 <strong>and</strong> HeLa cells using the phRL-TK Vector<br />
for a transfection control. Twenty-four hours post-transfection the cells were lysed with Passive Lysis 5X Buffer (Cat.# E1941) <strong>and</strong> luminescence<br />
was measured using the Dual-Luciferase® Reporter Assay System (Cat.# E1910). Relative light units were normalized to the Renilla luciferase<br />
expression from the phRL-TK Vector control. The fold increase in expression values is listed above each pair of bars. A repeat of this<br />
experiment yielded similar results.<br />
luc+<br />
PXR/CAR/RXR Pbx-1<br />
CCAAT<br />
SRF<br />
Gfl-1B<br />
Cut-like RFX1<br />
BRN-23 GBF3<br />
OBF1<br />
VIS1 En-1<br />
HEN1 SRF<br />
Sox-5<br />
NMP4<br />
NKX3.1 AREB6<br />
C-Myb<br />
SRF<br />
HNF1 AhR<br />
ATF4<br />
Otx2<br />
VIS1<br />
VIS 1<br />
Lmo2 NFY<br />
MyT1<br />
VIS 1<br />
ST/TA MEF<br />
Lmo2<br />
Mt<br />
TCF/LEF-1<br />
GFI1<br />
Pit1<br />
OBF 1<br />
E2F<br />
GSh-1<br />
MIBP-1/RFX1 POZ CDF-1 v-Myb<br />
FAST-1/S MAD<br />
Pbx1/Meis1<br />
MESI1<br />
COMP1<br />
E2F<br />
Pax-3<br />
Tst-1/Oct-6<br />
MEIS 1<br />
E2F Ikaros 3<br />
WHP<br />
CBF 3<br />
E4BP4 ST/TA<br />
CDE/CHR<br />
TCF/LEF-1<br />
MSX1/MSX-2 MyT1 P53<br />
C-Ets-1 GBF3<br />
AD-b<br />
HNF1<br />
Oct1,2<br />
GABP E2<br />
E4BP4<br />
NGN1/3<br />
v-Myb Brn3<br />
FOXF2<br />
PAR<br />
Pbx1<br />
TEF-1<br />
CCAAT<br />
OBF1<br />
C2HC SRE<br />
OBF1<br />
Cut-like<br />
FOXF2<br />
MEIS1 ST/TA<br />
Elk-1<br />
C2H2<br />
SOX-5<br />
GBF2 Pbx1<br />
MOK-2<br />
v-Myb POZ<br />
Brn2<br />
c-Ets-1<br />
MMC CCAATHIF1<br />
Pax1<br />
COMP1 VIS1<br />
PBX/MEIS1<br />
ST/TA<br />
ATF MEF2 E4BP4<br />
Tst-1/Oct-6<br />
SRF<br />
Brn23<br />
COMP1<br />
MOK-2 GATA<br />
Clox<br />
RF-7<br />
c-Myb TATA<br />
GBF1<br />
GLI-k<br />
POZ<br />
HNF6<br />
B-cell<br />
MEF<br />
TALE<br />
ZF<br />
Smad4 MIBP1 Pbx1<br />
Cut-like TCF/LEF-1<br />
Albumin D-box HEN1<br />
Lmo2<br />
RFX1<br />
v-Myb<br />
C-r<br />
CHOP CDE/CHR ST/TA<br />
HNF4 CREB<br />
ST/TA<br />
NKX3.1<br />
WHP<br />
Pax-6 Tax/CREB<br />
FLI<br />
FAST-1/SMAD NF-KB Se-CtRNA<br />
PXR/RXR/CAR<br />
MZF-1 Thing1<br />
AP 2<br />
MAZ BPV<br />
CREB<br />
c-Myb<br />
CREB<br />
NMP4<br />
COMP1<br />
GLI-K<br />
C-r<br />
Pbx1/Meis1<br />
PAR<br />
PAX2/5/8<br />
P53FLI<br />
E2F<br />
CREB<br />
HAND2<br />
NMP4 E2F E4BP4<br />
PAR<br />
Avian C<br />
MyT1 c-Myb<br />
TALE<br />
MOK-2<br />
Pbx-1 Pax-3 Cut-like<br />
NUR77/Nurr1/Nor-1<br />
Ikaros-3<br />
ST/TA<br />
Cut-like RAR<br />
CREB<br />
Elk-1<br />
Elk-1 E2FCREB<br />
PAR<br />
GFI1 E2F<br />
E2F C-Abl<br />
c-Myb v-Myb CDF-1 ST/TA<br />
SRF<br />
Figure 8.4. Reduced number of consensus transcription factor binding sites for the luc2 gene. The number of consensus transcription<br />
factor binding sites identified in the luc+ gene have been greatly reduced in the luc2 gene.<br />
Ideally, the reporter lifetime would be reduced to zero,<br />
completely eliminating the pooling of different<br />
transcriptional rates in each assay measurement. Only the<br />
transcription rate at the instant of the assay would be<br />
represented by the accumulation of reporter protein within<br />
the cells. Unfortunately, a zero lifetime would also yield<br />
zero accumulation, <strong>and</strong> thus no reporter could be measured.<br />
A compromise must be reached since, as lifetime reduces,<br />
<strong>Protocols</strong> & <strong>Applications</strong> <strong>Guide</strong><br />
www.promega.com<br />
rev. 3/09<br />
luc2<br />
v-Myb<br />
Bel-1<br />
3351MC07_1A<br />
11.8<br />
HeLa<br />
NF-kB<br />
4731MA<br />
p53<br />
LBP1c/LSF<br />
v-Myb<br />
p53<br />
PAX9<br />
VDR/RXR<br />
FKHRL1<br />
so does the amount of reporter available for detection in<br />
the assay. This is where the high sensitivity of luminescent<br />
assays is useful. Relative to other reporter technologies, the<br />
intracellular stability of luciferase reporters may be greatly<br />
reduced without losing measurable signals. Thus, the high<br />
sensitivity of luciferase assays permits greater dynamics<br />
in the luciferase reporters.<br />
PAX9<br />
c-Myb<br />
CDF1<br />
Baso<br />
p53<br />
4760MA<br />
PROTOCOLS & APPLICATIONS GUIDE 8-6
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