Specific, rapid, and convenient analysis of the in in vivo activation of transcriptional pathways.
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. The commonly used plasmid-based transcriptional reporter vectors containing different reporter genes can be delivered by transient transfection to the nucleus of target cells to monitor the activation of signal transduction pathways converging at a specific response element.
Lentiviral expression vectors are the most effective vehicles for delivering genetic material to almost any mammalian cell—including non-dividing cells and to model organisms. As with standard plasmid vectors, it is possible to introduce lentiviral Transcriptional Reporter (TR) constructs in plasmid form into the cells with low-to-medium efficiency using conventional transfection protocols. However, by packaging the lentiviral TR vector construct in pseudoviral particles, you can obtain highly efficient transduction and heritable expression of transcriptional reporter constructs—even with the most difficult-to-transfect cells, like primary, stem, and differentiated cells. In comparison to retroviral delivery systems, lentivectors enter the cell nucleus without requiring cell replication.
Create your own reporter cell line with SBI’s ready-made lentivirus or choose from SBI’s clonal reporter cell lines.
Clonal cell lines isolated and characterized for accurate transcriptional reporting using lentiviral expression vectors. As with standard plasmid vectors, it is possible to introduce lentiviral Transcriptional Reporter (TR) constructs in plasmid form into the cells with low-to-medium efficiency using conventional transfection protocols. However, by packaging the lentiviral TR vector construct in pseudoviral particles, you can obtain highly efficient transduction and heritable expression of transcriptional reporter constructs—even with the most difficult-to-transfect cells, like primary, stem, and differentiated cells. In comparison to retroviral delivery systems, lentivectors enter the cell nucleus without requiring cell replication.
NF-κB/293/GFP-Luc™ Transcriptional Reporter Cell Line
Monoclonal human embryonic kidney (HEK)-293-based cell line for analysis of Nuclear Factor kappa B (NF-κB) pathway activation featuring GFP and Luciferase dual reporter system (cat# TR860A-1).
Highly sensitive reporter cell line - first of its kind with Dual reporters for GFP and Firefly Luciferase
Monoclonal HEK-293 cell line with stably integrated lentiviral transcriptional reporter vector with 300-fold NF-κB-dependent activation
Monitor transactivation in live cells with GFP
Quantitate NF-κB response using Luciferase
The NF-κB/293/GFP-Luc™ cell line was specifically designed for monitoring the NF-κB signal transduction pathway in vitro. The unique features of this reporter cell line allows researchers to monitor the NF-κB pathway by the detection of GFP fluorescence as well as Luciferase for quantitative transcription activation reporter assays. The NF-κB/293/GFP™ cells were derived from System Biosciences’ 293 TN Producer Cell Line (Cat. # LV900A-1). 293 TN cells were transduced with HIV-based pseudoviral particles packaged with a lentivector that co-expresses destabilized copGFP and Firefly Luciferase reporters coupled with the minimal cytomegalovirus (mCMV) promoter in conjunction with four copies of the NF-κB consensus transcriptional response elements.
Monitor NF-κB Activation in real-time with GFP
Quantitate NF-κB Activation with Luciferase Assays
Highly sensitive cell line responds to TNFα as seen with the GFP FACS data above. The GFP fluorescence saturates and the Luciferase quantitation assays clearly reveal a linear induction relationship with increasing amounts of TNFα.
NF-κB
NF-κB, a member of the rel family of transcription factors, regulates several important physiological processes, including immune responses, inflammation, cell growth, apoptosis, tumorigenesis, and the expression of certain virus genes (HIV and CMV). As a result, the NF-κB signaling pathway has been a target for pharmacological intervention, especially in models of inflammation or cancer, where the pathway is often constitutively active (1). Over 750 inhibitors of the NF-κB pathway have been identified, including both natural and synthetic molecules (1). Conversely, many different stimuli have been identified which activate the NF-κB pathway, including cytokines such as TNF-α and interleukin-1β, pathogenic bacteria and viruses, bacterial lipopolysaccharide and peptidoglycan, and oxidative stress. The NF-κB/293/GFP™ cell line allows the researcher to study both potential inhibitors and activators of the NF-κB pathway, and as such, is an extremely valuable tool for a wide variety of research applications.
NF-κB/293/GFP™ Transcriptional Reporter Cell Line
A human embryonic kidney (HEK)-293-based cell line for analysis of Nuclear Factor kappa B (NF-κB) pathway activation
Clonal HEK-293 cell line with stably integrated lentiviral transcriptional reporter vector with 300-fold NF-κB-dependent activation of GFP reporter gene—3 times more sensitive than competitor cell lines
Study mechanism of signal transduction in the NF-κB pathway
Screen for compounds that inhibit or activate the NF-κB pathway
RNAi screening for genes involved in inhibition or activation of the NF-κB pathway
Green Fluorescent Protein (GFP) reporter gene allows for ease of detection and Fluorescence Activated Cell Sorting (FACS)
System Biosciences (SBI) has developed a stable HEK-293-based NF-κB reporter cell line, NF-κB/293/GFP™, for the study of the NF-κB signal transduction pathway. Stimulation of the NF-κB pathway results in up to a 300-fold increase in expression of the reporter gene, GFP, in this unique cell line (Figure 1). Utilization of the GFP reporter gene allows the researcher to detect NF-κB activation by fluorescent microscopy, and offers the advantage of allowing for GFP-positive or negative cells to be sorted by FACS. As a result, the NF-κB/293/GFP™ cell line is completely compatible with SBI's HIV- and FIV-based genome-wide siRNA libraries and individual siRNA lentiviral vectors for RNAi knockdown studies, as well as cDNA expression vectors to identify genes involved in the stimulation or inhibition of the NF-κB pathway. The NF-κB/293/GFP™ cell line was developed by transduction of HEK-293 cells with a lentiviral transcriptional reporter vector carrying four copies of the consensus NF-κB transcription factor binding site located upstream of the minimal cytomegalovirus (mCMV) promoter which regulate NF-κB-dependent expression of GFP (Figure 2), followed by selection of a clonal population that maintained stable chromosomal integration of the lentiviral vector provirus and responded strongly to stimuli known to activate the NF-κB pathway. The result is a cell line that has an extremely robust response to NF-κB stimuli such as tumor necrosis factor-α and interleukin-1β (TNF-α and IL-β respectively; Figure 1). NF-κB/293/GFP™ cells are highly transfectable and transducible human cell lines which serve as useful in vitro cell models for a variety of research applications, including screening of small molecule inhibitors or activators of the NF-κB pathway, and the identification of genes involved in the inhibition or activation of the pathway by use of genome-wide siRNA libraries or specific siRNAs, available from System Biosciences.
NF-κB
NF-κB, a member of the rel family of transcription factors, regulates several important physiological processes, including immune responses, inflammation, cell growth, apoptosis, tumorigenesis, and the expression of certain virus genes (HIV and CMV). As a result, the NF-κB signaling pathway has been a target for pharmacological intervention, especially in models of inflammation or cancer, where the pathway is often constitutively active (1). Over 750 inhibitors of the NF-κB pathway have been identified, including both natural and synthetic molecules (1). Conversely, many different stimuli have been identified which activate the NF-κB pathway, including cytokines such as TNF-α and interleukin-1β, pathogenic bacteria and viruses, bacterial lipopolysaccharide and peptidoglycan, and oxidative stress. The NF-κB/293/GFP™ cell line allows the researcher to study both potential inhibitors and activators of the NF-κB pathway, and as such, is an extremely valuable tool for a wide variety of research applications.
NF-κB/Jurkat/GFP Transcriptional Reporter Cell Line
A human T lymphocyte-based cell line for analysis of Nuclear Factor kappa B (NF-κB) pathway activation
Clonal Jurkat cell line with stably integrated lentiviral transcriptional reporter vector with 30-fold NF-κB-dependent activation of GFP reporter gene
Study mechanisms of signal transduction in the NF-κB pathway in a physiologically relevant cell line
Suitable for high-throughput screening for compounds that inhibit or activate the NF-κB pathway
Perform RNAi screening for genes involved in inhibition or activation of the NF-κB pathway
Green Fluorescent Protein (GFP) reporter gene allows for ease of detection and Fluorescence Activated Cell Sorting (FACS)
System Biosciences (SBI) has developed a stable T lymphocyte-based NF-κB reporter cell line, NF-κB/Jurkat/GFP™, for the study of the NF-κB signal transduction pathway. The NF-κB/Jurkat/GFP™ cell line was developed by introduction of a transcriptional reporter vector carrying four copies of the consensus NF-κB transcription factor binding site located upstream of the minimal cytomegalovirus (mCMV) promoter (Figure 1). A clonal population was then selected which exhibited low background levels of GFP expression but which responded strongly to TNF-α, a known stimulus of the NF-κB pathway.
Stimulation of the NF-κB pathway results in up to a 30-fold increase in expression of the reporter gene, GFP, in this unique cell line (Figure 2). Utilization of the GFP reporter gene allows the researcher to detect NF-κB activation by fluorescent microscopy, and offers the advantage of allowing for GFP-positive or negative cells to be sorted by FACS. As a result, the NF-κB/Jurkat/GFP™ cell line is completely compatible with SBI's HIV- and FIV-based genome-wide siRNA libraries and individual siRNA lentiviral vectors for RNAi knockdown studies, as well as cDNA expression vectors to identify genes involved in the stimulation or inhibition of the NF-κB pathway.
The result is a cell line that has an extremely robust response to NF-κB stimuli such as tumor necrosis factor-α. NF-κB/Jurkat/GFP™ cells are a transducible human cell line which serve as useful in vitro models for a variety of research applications, including screening of small molecule inhibitors or activators of the NF-κB pathway, and the identification of genes involved in the inhibition or activation of the pathway by use of genome-wide siRNA libraries or specific siRNAs, available from System Biosciences.
NF-κB
NF-κB, a member of the rel family of transcription factors, regulates several important physiological processes, including immune responses, inflammation, cell growth, apoptosis, tumorigenesis, and the expression of certain virus genes (HIV and CMV). As a result, the NF-κB signaling pathway has been a target for pharmacological intervention, especially in models of inflammation or cancer, where the pathway is often constitutively active (1). Over 750 inhibitors of the NF-κB pathway have been identified, including both natural and synthetic molecules (1). Conversely, many different stimuli have been identified which activate the NF-κB pathway, including cytokines such as TNF-α (Figure 3 below) and interleukin-1β, as well as pathogenic bacteria, viruses, bacterial lipopolysaccharide and peptidoglycan.
Figure 3. Activation of NF-B/Jurkat/GFP™ cells with increasing amounts of TNF-α.
NF-B/Jurkat/GFP™ Reporter cells (5x10^5 cells) were plated at a concentration of 1 million cells/ml into each well of a 24-well plate. TNF-α was added in the amount indicated in the Figure. After 24 hours, 100 μl of the cells were transferred to a well of a Costar® UV plate (96 well, No lid, w/ UV Transparent Flat Bottom, Corning, NY, Cat# 3635) and the intensity of GFP fluorescence was measured (Excitation 485+/-20, Emission 528+/-20) in a Synergy™ HT Multi-Detection Microplate Reader (BioTek, Winooski, Vermont). The intensities of GFP measured were plotted against the amount of TNF-α (A). The fluorescent cells in the original 24-well plate were also photographed on a Zeiss inverted epi-fluorescence microscope (B). Alternatively, 200 ul of the cells were fixed with 200 μl of 4% formaldehyde and GFP reporter induction analyzed by flow cytometry, and either the GFP intensities (C) or the percentage of GFP positive cells (D) were plotted against the amount of TNF-α.
The NF-κB/Jurkat/GFP™ reporter cell line can also be stimulated through its T-Cell Receptor, for example using anti-CD3/CD28 beads (Figure 4 below), and responds with appropriate TcR-mediated signaling.
SBI's NF-κB/Jurkat/GFP™ reporter cell line allows the high-throughput analysis of both potential inhibitors and activators of the NF-κB pathway, making it an extremely valuable tool for a wide variety of research applications.
