The tetracycline-On (Tet-On) system is a conditional gene expression tool that activates target gene expression in the presence of tetracycline or doxycycline. This system utilizes genetically engineered mice with a liver-specific promoter driving the expression of the reverse tetracycline-controlled transcriptional activator (rtTA).
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 binds to the rtTA, causing conformational change and increased affinity for the tetO DNA binding site. The tetR DNA-binding domain of doxycycline-bound rtTA binds to the tetO sequence of the TRE element. The VP16 domain further recruits RNA polymerase, leading to the transcription, forming mRNA, and subsequent expression of the downstream reporter target gene.
For experiments of less than 10 days, continuously administer doxycycline via drinking water using the following protocol. First, dissolve 5 grams of sucrose in 100 milliliters of tap water, and autoclave.
Then, dissolve 100 milligrams of doxycycline-hyclate in 5 milliliters of the sucrose solution in a 15-milliliter conical tube. Next, use a 10-milliliter syringe to sterile filter the solution through a 0.2-micron filter, then, add the filtered doxycycline solution to the remaining 95 milliliters of sucrose solution, and supply the solution to the mouse instead of drinking water. Check daily and replace immediately if the solution becomes cloudy as this indicates bacterial overgrowth.
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