Name: Inducible Tet-On Regulatable System-Based Gene Expression In Vivo: A Tetracycline-Induced Gene Expression System for Target Gene Transcription Activation in Mouse Model
Uploaded: 2023-04-30T14:56:22
Description: Source: Hubner, E. K. et al.&#160; Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein ...

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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:&#160;Depending on the type and duration of the experiment, doxycycline can be supplied in drinking water.
<ol>
	<li>For short term experiments (&#60;10 days) administer doxycycline via drinking water using the following protocol.
	<ol>
		<li>Dissolve 5 g sucrose in 100 mL of tap water. Autoclave.</li>
		<li>Dissolve 100 mg of doxycycline-hyclate in 5 mL of sucrose solution (step 1.1.1) in a 15-mL conical tube.</li>
		<li>Using a 10-mL syringe, sterile filter the solution through a 0.2 &#181;m filter. Add to the sucrose solution prepared in step 1.1.1.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>

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			<td>D(+)-Saccharose</td>
			<td>Carl Roth</td>
			<td>4621.1</td>
			<td>For sweetening of the doxycyline solution</td>
		</tr>
		<tr>
			<td>Doxycycline hyclate</td>
			<td>AppliChem</td>
			<td>A2951,0025</td>
			<td>Activation of tetracycline-dependent expression</td>
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			<td>Injekt 10 ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td></td>
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			<td>Filtropur S 0.2</td>
			<td>Sarstedt</td>
			<td>831.826.001</td>
			<td>For filtration of doxycycline</td>
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inducible tet-on regulatable system-based gene expression in vivo: a tetracycline-induced gene expression system for target gene transcription activation in mouse model

Source: Hubner, E. K. et al.&#160; <a href="https://www.jove.com/v/56613">Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection.</a> 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.

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

Source: Hubner, E. K. et al.&#160; Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein ...

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.

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2.2K Views. 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. 

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

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

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Genetics

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Developmental Biology

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...