Use the Tet-On or Tet-Off systems depending on your desired gene expression. The Tet-On system activates gene expression in the presence of tetracycline or its analogs, while Tet-Off represses gene expression. Choose carefully; the system’s functionality directly impacts your experimental results.
Tet-On: This system utilizes a reverse tetracycline transactivator (rtTA) that binds to the tetracycline response element (TRE) only when tetracycline is present. Therefore, gene expression linked to the TRE is induced upon tetracycline addition.
Consider using doxycycline instead of tetracycline. Doxycycline offers better cell permeability and a longer half-life, resulting in more tightly controlled gene expression.
Tet-Off: Conversely, the Tet-Off system uses a tetracycline transactivator (tTA) that constitutively binds to the TRE. Adding tetracycline disrupts this binding, switching off gene expression. This system provides tight basal repression.
Optimize tetracycline concentration. Perform titration experiments to determine the optimal concentration that achieves the desired level of gene expression or repression without affecting cell viability. Incorrect dosage could lead to leaky expression or toxicity.
Monitor gene expression regularly. Employ quantitative PCR (qPCR) or Western blotting to verify the effectiveness of your chosen system and confirm the desired level of inducibility. This helps validate your experimental design and troubleshooting.
For best results, use a well-characterized and commercially available Tetracycline Inducible Promoter system. These systems often include optimized vectors and detailed protocols. This minimizes setup time and reduces potential experimental error.
