The Xenobiotic response element (XRE) Luciferase Reporter Lentiviruses are replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles that are ready to transduce most types of mammalian cells, including primary and non-dividing cells. The particles contain a firefly luciferase gene driven by three copies of an XRE located upstream of the minimal TATA promoter (Figure 1), and an antibiotic selection gene (puromycin) for the selection of stable clones. After transduction, the activation of aryl hydrocarbon receptor (AhR) in the target cells can be monitored by measuring the luciferase activity.
Figure 1. Schematic of the lenti-vector used to generate the XRE luciferase reporter lentivirus.
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Activation of XRE luciferase reporter activity in HepG2 cells
The lentivirus particles were produced from HEK293T cells. They are supplied in cell culture medium containing 90% DMEM + 10% FBS.
Background
The aryl hydrocarbon receptor (AhR) functions as a sensor of xenobiotic chemicals, notably aromatic hydrocarbons, natural plant flavonoids, and plant polyphenolics, and it regulates stress pathways in eukaryotic cells. It is a cytosolic transcription factor that is normally kept inactive by binding to co-chaperones such as heat shock protein 90 (HSP90). Upon binding to xenobiotic chemicals, the chaperone dissociates from AhR, which results in AhR translocating into the nucleus and dimerizing with AhR nuclear translocator (ARNT). The heterodimer binds to a canonical XRE, regulating the transcription of target genes.
Storage/Stability
Lentiviruses are shipped with dry ice. For long-term storage, it is recommended to store the lentiviruses at -80°C.
Applications
Screen for activators or inhibitors of the AhR signaling pathway.
Generate a XRE luciferase reporter stable cell line (puromycin resistant) following puromycin selection and limiting dilution
Shipping Temperature
-80°C
Notes
To generate the XRE luciferase reporter stable cell line, remove the growth medium 48 hours after transduction and replace it with fresh growth medium containing the appropriate amount of puromycin for antibiotic selection of transduced cells. To determine the concentration of puromycin needed for your cell line, perform a kill curve (for more information, visit our Resources page, frequently asked questions: what is a kill curve?).
The following Lentivirus Reporter Controls are available from BPS Bioscience to meet your experimental needs:
Negative Control Luciferase Lentivirus (BPS Bioscience #79578): Ready-to-transduce lentiviral particles expressing firefly luciferase under the control of a minimal promoter. The negative control is important to establish the specificity of any treatments and to determine the background reporter activity.
Renilla Luciferase Lentivirus (BPS Bioscience #79565): Ready-to-transduce lentiviral particles expressing Renilla luciferase under the CMV promoter. The Renilla Luciferase lentivirus can serve as an internal control to overcome sample-to-sample variability when performing dual-luciferase reporter assays.
Firefly Luciferase Lentivirus (BPS Bioscience #79692-G, #79692-H, #79692-P): Ready-to-transduce lentiviral particles expressing firefly luciferase under the CMV promoter. It serves as a positive control for transduction optimization studies.
Biosafety The lentiviruses are produced with SIN (self-inactivation) lentivector which ensures self-inactivation of the lentiviral construct after transduction and integration into the genomic DNA of the target cells. None of the HIV genes (gag, pol, rev) will be expressed in the transduced cells, as they are expressed from packaging plasmids lacking the packing signal and are not present in the lentivirus particle. Although the pseudotyped lentiviruses are replication-incompetent, they require the use of a Biosafety Level 2 facility. BPS Bioscience recommends following all local federal, state, and institutional regulations and using all appropriate safety precautions.
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