GAL4-DBD-AR-LBD-BSD

The GAL4-DBD-AR-LBD-BSD construct is designed to measure the activity of the Androgen Receptor (AR), a ligand-dependent nuclear receptor that plays a critical role in regulating genes associated with the development and maintenance of reproductive and muscle tissues, as well as metabolic pathways. This construct incorporates a fusion of the GAL4 DNA-binding domain (DBD), the AR ligand-binding domain (LBD), and a BSD selection marker. The GAL4 DBD targets a specific DNA sequence in the 5x GAL4 UAS-TAG-Puro construct, which contains a reporter gene (GFP) upstream of the UAS sequence. When the AR-LBD is activated by its ligand (testosterone), the GAL4-DBD-AR-LBD fusion protein binds to the UAS sequence, resulting in transcriptional activation of the reporter gene. This setup provides a straightforward and measurable readout of AR activity via GFP fluorescence.

For added versatility in reporter detection, the GAL4-DBD-AR-LBD-BSD construct can be paired with the GAL4-TAR-Puro construct (which uses RFP as the reporter) or the GAL4-TAL-Puro construct (utilizing Firefly Luciferase as the reporter), depending on the experimental needs.

Experimental Data and Interpretation

HEK293 cells were co-transfected with GAL4-DBD-AR-LBD-BSD and GAL4-TAG-Puro constructs. After 16 hours, cells were treated with either DMSO (control) or 10 nM testosterol (ligand) in 10% FBS culture medium and incubated for an additional 16 to 24 hours. The experimental results under each condition demonstrate AR’s behavior:

• DMSO (Control): Cells treated with DMSO show baseline GFP fluorescence, indicating minimal or no transcriptional activation in the absence of ligand. This serves as a control reference level to compare against ligand-induced activity.
• 10nM Testosterone (Ligand): Treatment with testosterone activates the AR-LBD, promoting its binding to the UAS sequence and leading to robust GFP expression. This heightened fluorescence demonstrates the ligand-dependent transcriptional activation of the reporter gene, highlighting the construct’s responsiveness to androgenic signals.

This clear ligand-dependent fluorescence provides a reliable indicator of AR activity and validates the effectiveness of the GAL4-DBD-AR-LBD system in detecting androgen receptor activation.

Application and Research Utility

This construct offers researchers a highly specific tool for studying the activity of the Androgen Receptor (AR) in real-time. By monitoring GFP fluorescence in response to testosterone or other androgenic ligands, researchers can investigate AR’s role in processes related to reproductive biology, muscle development, and metabolism. The ability to modulate and measure AR activity is invaluable for exploring signaling pathways involved in androgen-dependent disorders, prostate cancer, and therapeutic research focused on androgen receptor antagonists.

This product is supplied as pre-packaged lentiviral particles only. Plasmids are not available for this product.