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.