Eukaryotic gene expression is regulated by a wide variety of developmental and environmental stimuli. First, an extracellular signaling molecule binds to a specific receptor. The signal is then transmitted through a series of molecular cascades, which activate or deactivate specific transcription factors (TFs) that regulate gene expression. The expression of any given gene is controlled by multiple transcription factors, which in turn are modulated by multiple signal transduction pathways. Many of these signal transduction pathways converge at transcription factors that bind to specific transcriptional response elements (TREs) found in the promoters of various genes and modulate the transcription of these genes. The activation of a signal transduction pathway (e.g. by growth factors, drugs, etc.) can therefore be monitored by the expression level of the reporter gene controlled by a promoter containing these response elements.
Commonly used plasmid-based transcriptional reporter vectors can often skew transcriptional network reporting due to their episomal nature. SBI's lentivector-based transcription reporters integrate into the host's genome and enable proper chromatinization to produce more faithful transcriptional activity reporting.
Advantages of lentivector technology include:
Ready-to-use pre-packaged constructs with a wide range of Transcriptional Response Elements (TREs) for multiple transcriptional factors.
Lentiviral reporter constructs can efficiently transduce nearly all cell types, even those that are difficult-to-transfect such as primary or non-dividing mammalian cells.
Our lentiviral-based reporter system is a novel approach to study transcriptional regulation and offers many advantages over current transcription reporter systems. TR constructs will integrate into the genome and therefore be subject to chromatin regulation (Leung, T.H., et.al., 2004). Expression of the reporter gene indicates activation of a given transcriptional response element (TRE) by the cognate transcriptional factor in the natural chromosomal environment rather than in the episomal state in the nucleoplasm as is the case for conventional plasmid-based TR vectors. Tandem copies of integration can be avoided, thus allowing for faithful promoter regulation. Copy number of reporter constructs can be controlled by varying the multiplicity of infection (MOI).
Construction of stable reporter cell lines is possible with TR lentivectors in just several days without the need for conventional, low efficiency selection of stable transfectants.
Monitoring of signaling pathways by flow cytometry (FACS) is enabled by GFP reporters or quantitate transactivation with Luciferase.
pGreenFire1 (pGF1) is a versatile HIV-based lentivector that co-expresses destabilized copGFP and Firefly Luciferase enabling the detection of both GFP signals as well as Luciferase for quantitative transcription activation reporter assays.
Lentivector map of pGreenFire1
The pGreenFire pathway reporters are also available with an optional EF1-Puro or EF1-Neo selection cassette placed downstream of the WPRE element. To include this option on your pGreenFire vector order, simply place a "-P" for Puro or "-N" for Neo to designate which option you want. The selection cassettes are no extra charge. Example: pGreenFire1-HIF1(plasmid, catalog #TR026PA-1, would be #TR026PA-P to include the EF1-Puro selection cassette.
Measure both GFP and Luciferase with pGreenFire1
Features and Benefits of pGreenFire1 (pGF1) Lentivectors
pGreenFire1 is a dual reporter vector and so has the advantage of being able to determine whether reporter activation has taken place by assessing both GFP and firefly luciferase levels.
Firefly luciferase provides a familiar, well-established and quantitative assessment of transcription activation, while the GFP reporter enables non-destructive live cell imaging.
Proviral reporters are integrated into normal chromatin and therefore provide a more accurate model for the study of transactivation and epigenetic effects.
pGreenFire1 lentiviral reporters allow researchers to quickly and easily establish stable reporter cell lines using either their own transcriptional response elements or any of SBI’s pre-made, pre-validated reporters.
Stable reporter cell lines eliminate the need to transfect reporter plasmids for each experiment and so reduces the well-to-well variability often seen with plasmid reporter transfection.
No need to have a second reporter (eg Renilla luciferase or beta-galactosidase) to normalize transfection levels.
Minimal CMV (mCMV) basal promoter has extremely low activity in the absence of transcription response elements.
List of pre-made GreenFire Pathway Reporters
Pathway
Transcription Factor
Plasmid Catalog #
Virus Catalog #
TRE sequence (repeated 4x)
Negative control
none
TR010PA-1
TR010VA-1
Positive control
CMV
TR011PA-1
TR011VA-1
C/EBP
C/EBPa
TR040PA-1
TR040VA-1
CCATTGCAAAATCAGCT
cAMP/PKA
CREB
TR035PA-1
TR035VA-1
TGACGTCACGGCATTGTAGCG
p53/DNA Damage
p53
TR025PA-1
TR025VA-1
CGACATGCCCGGGCATGT
Hypoxia
HIF-1
TR026PA-1
TR026VA-1
CTACGTGCTCGGCATTGTAG
Type I Interferon
ISRE
TR016PA-1
TR016VA-1
CAGTTTCACTTTCCCTTT
Interferon Gamma
STAT1
TR015PA-1
TR015VA-1
CATGTTATGCATATTCCTGTAAGTG
Interferon Gamma
GAS
TR027PA-1
TR027VA-1
AGTTTCATATTACTCTAAATC
MAPK/ERK
Elk-1/SRF
TR029PA-1
TR029VA-1
ATGCCCATATATGGTAAT
MAPK/JNK
AP1
TR014PA-1
TR014VA-1
TTCCGGTGACTCAGTCAAGCG
MEF2
MEF2
TR030PA-1
TR030VA-1
CTATTTATAGaCTATTTATAG
NFkB
NF- kB
TR012PA-1
TR012VA-1
GGGGACTTTCC
Notch
RBP-Jk
TR020PA-1
TR020VA-1
GTGGGAACGGCATTGTAGCG
PKC/Ca++
NFAT
TR034PA-1
TR034VA-1
AGAGGAAAATTGCTGATTGCA
Retinoic Acid Receptor
Retinoic Acid Receptor (RAR)
TR037PA-1
TR037VA-1
AGGTCACCTGGAGGTCACGG
SP1
SP1
TR036PA-1
TR036VA-1
AGGGGGCGGGGT
Wnt
TCF/LEF1
TR013PA-1
TR013VA-1
CCTTTGATCTTACC
Wnt
cJun
TR033PA-1
TR033VA-1
TGGTGTAATAC
Sterol Response
LXRE
TR018PA-1
TR018VA-1
GGGTTACTGGCGGTCATTGTA
Sterol Response
SREBP
TR028PA-1
TR028VA-1
ATCACGTG
Stem Cells
Oct4
TR039PA-1
TR039VA-1
TGTCGAATGCAAATCACTAGAA
Stem Cells
Nanog
TR019PA-1
TR019VA-1
ACCCTTCGCCGATTAAGTACTTAAG
Oculogenesis
Pax6
TR031PA-1
TR031VA-1
TCACGC
Adipocyte Differentitaion (Sterol Response)
PPARy
TR032PA-1
TR032VA-1
TGTAGGTCACGGTGACCTAC
TGFbeta
SMAD
TR038PA-1
TR038VA-1
AGCCAGACATCGCATCAGCA
Yeast reporter
GAL4
TR017PA-1
TR017VA-1
GGAGCACTGTCCTCCGAACGTC/TC- GGAGAACTGTCGTCCGAACG
SBI’s pGreenFire lentivectors enable the simultaneous tracking of GFP and the quantitative measurement of Luciferase reporters.
Sample data using the GreenFire dual reporters
Sample Data using the pGreenFire1-NF-κB Transcription Reporter
Monitoring NF−κB transactivation using GFP epifluorescence.
293TN cells were transduced with the packaged pGF1-NFκB reporter virus. Two days later, the cells were split into 96 well plates and then treated with the indicated amount of TNF-α. Levels of NFκB activation was assessed 18 hours post-induction using epifluorescence microscopy.
Quantitative analysis of NF-κB transactivation using Firefly Luciferase
293TN cells were transduced with either the packaged pGF1-NFκB or pGF1-mCMV (negative control) reporter virus. Two days later, the cells were split into 96 well plates and then treated with the indicated amount of TNF-α. Levels of NFκB activation was quantitated 18 hours post-induction using a standard firefly luciferase assay (Promega).
