Inducible Tet-On Regulatable System-Based Gene Expression In Vivo: A Tetracycline-Induced Gene Expression System for Target Gene Transcription Activation in Mouse Model
Abstract
Source: Hubner, E. K. et al. Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection. J. Vis. Exp. (2018)
This video demonstrates the tetracycline, or Tet-On, gene expression system in a mouse model. In the presence of doxycycline, a tetracycline derivative, the Tet-On system consisting of reverse tetracycline-controlled transactivator protein becomes activated and expresses the gene of interest.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Induction of Tetracycline-dependent Gene or shRNA Expression
CAUTION: Doxycycline may be harmful. Please refer to the safety data sheet.
NOTE: Depending on the type and duration of the experiment, doxycycline can be supplied in drinking water.
- For short term experiments (<10 days) administer doxycycline via drinking water using the following protocol.
- Dissolve 5 g sucrose in 100 mL of tap water. Autoclave.
- Dissolve 100 mg of doxycycline-hyclate in 5 mL of sucrose solution (step 1.1.1) in a 15-mL conical tube.
- Using a 10-mL syringe, sterile filter the solution through a 0.2 µm filter. Add to the sucrose solution prepared in step 1.1.1.
- Supply doxycycline-sucrose solution as drinking water to the mouse. Check daily and replace when the solution becomes cloudy indicating bacterial overgrowth (replace clear solution after three days at the latest).
開示
The authors have nothing to disclose.
Materials
| D(+)-Saccharose | Carl Roth | 4621.1 | For sweetening of the doxycyline solution |
| Doxycycline hyclate | AppliChem | A2951,0025 | Activation of tetracycline-dependent expression |
| Injekt 10 ml Syringe | Braun | 4606108V | |
| Filtropur S 0.2 | Sarstedt | 831.826.001 | For filtration of doxycycline |
