Inducible Tet-On Regulatable System-Based Gene Expression In Vivo: A Tetracycline-Induced Gene Expression System for Target Gene Transcription Activation in Mouse Model

The tetracycline-On or Tet-On system is a conditional gene expression system that activates expression of gene of interest in the presence of tetracycline or its analog, doxycycline.

Begin with a genetically engineered mouse with a modified genome containing gene encoding reverse tetracycline-controlled transcriptional activator protein, or rtTA, driven by a liver-specific promoter in the liver cells or hepatocytes. Additionally, the mouse contains a gene of interest under the control of tetracycline operator or tetO sequences located upstream and selectively fused to a minimal promoter. The tetO and promoter are called the tetracycline-responsive element, or TRE.

The expressed rtTA - a fusion protein comprising the tet repressor, tetR, and viral protein VP16 transactivation domain - forms dimers, which are inactive and cannot bind to tetO sequence of the TRE, preventing the activation of target gene expression.

Prepare a solution containing doxycycline and sucrose. Sucrose addition increases palatability. After filtering, provide the doxycycline-sucrose solution as drinking water to the mouse. The administered doxycycline accumulates in the liver.

In the hepatocytes, doxycycline b