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

протокол

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.

1. For short term experiments (<10 days) administer doxycycline via drinking water using the following protocol.
   1. Dissolve 5 g sucrose in 100 mL of tap water. Autoclave.
   2. Dissolve 100 mg of doxycycline-hyclate in 5 mL of sucrose solution (step 1.1.1) in a 15-mL conical tube.
   3. 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.
   4. 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).

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Материалы

Name	Company	Catalog Number	Comments
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