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An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice.

Tillack K, Aboutalebi H, Kramer ER - PLoS ONE (2015)

Bottom Line: The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson's disease (PD) and drug addiction.Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification.We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre.

View Article: PubMed Central - PubMed

Affiliation: Development and Maintenance of the Nervous System, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT

Background & aims: The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson's disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification.

Methods & results: Here, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre. In combination with mice encoding tet-responsive luciferase, we visualized the DA system in living mice progressively over time.

Conclusion: These experiments establish TH-tTA and TH-rtTA mice as a powerful tool to generate and monitor mouse models for DA system diseases.

No MeSH data available.


Related in: MedlinePlus

Schematic overview of utilized transgenic mice and recombinant AAV (rAAV) constructs.TH-tTA and TH-rtTA mice express, under the tyrosine hydroxylase (TH) promoter, the tetracycline-regulated transactivator protein (tTA) or the reverse tetracycline-regulated transactivator protein (rtTA) in dopaminergic (DA) neurons. tTA binds to the tetracycline operon (tetO) in the absence of doxycycline (DOX) and rtTA binds in the presence of DOX, driving transient expression of gene X, for example the fluorescent protein Venus from a recombinant AAV vector (AAV-tetO-Venus), or luciferase and Cre from the LC1 construct. The LC1 encodes a luciferase enzyme, which can be used for bioluminescence in vivo imaging, and the Cre recombinase for deleting gene Y or removing floxed STOP codons (LSL). Cre is used here as a genetic switch to activate, from the ROSA locus, either expression of the reporter gene lacZ (encoding β-galactosidase) to visualize transgene expression or to activate expression of diphtheria toxin protein A (dt-a) to selectively induce cell death of DA neurons. Here we use Cre also to activate expression of the fluorescent protein mCherry from AAV-LSL-mCherry.
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pone.0136203.g006: Schematic overview of utilized transgenic mice and recombinant AAV (rAAV) constructs.TH-tTA and TH-rtTA mice express, under the tyrosine hydroxylase (TH) promoter, the tetracycline-regulated transactivator protein (tTA) or the reverse tetracycline-regulated transactivator protein (rtTA) in dopaminergic (DA) neurons. tTA binds to the tetracycline operon (tetO) in the absence of doxycycline (DOX) and rtTA binds in the presence of DOX, driving transient expression of gene X, for example the fluorescent protein Venus from a recombinant AAV vector (AAV-tetO-Venus), or luciferase and Cre from the LC1 construct. The LC1 encodes a luciferase enzyme, which can be used for bioluminescence in vivo imaging, and the Cre recombinase for deleting gene Y or removing floxed STOP codons (LSL). Cre is used here as a genetic switch to activate, from the ROSA locus, either expression of the reporter gene lacZ (encoding β-galactosidase) to visualize transgene expression or to activate expression of diphtheria toxin protein A (dt-a) to selectively induce cell death of DA neurons. Here we use Cre also to activate expression of the fluorescent protein mCherry from AAV-LSL-mCherry.

Mentions: LC1 mice [21], Rosa26R reporter mice [22] and ROSA26-dt-a mice [23] were described previously (Figs 1–6).


An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice.

Tillack K, Aboutalebi H, Kramer ER - PLoS ONE (2015)

Schematic overview of utilized transgenic mice and recombinant AAV (rAAV) constructs.TH-tTA and TH-rtTA mice express, under the tyrosine hydroxylase (TH) promoter, the tetracycline-regulated transactivator protein (tTA) or the reverse tetracycline-regulated transactivator protein (rtTA) in dopaminergic (DA) neurons. tTA binds to the tetracycline operon (tetO) in the absence of doxycycline (DOX) and rtTA binds in the presence of DOX, driving transient expression of gene X, for example the fluorescent protein Venus from a recombinant AAV vector (AAV-tetO-Venus), or luciferase and Cre from the LC1 construct. The LC1 encodes a luciferase enzyme, which can be used for bioluminescence in vivo imaging, and the Cre recombinase for deleting gene Y or removing floxed STOP codons (LSL). Cre is used here as a genetic switch to activate, from the ROSA locus, either expression of the reporter gene lacZ (encoding β-galactosidase) to visualize transgene expression or to activate expression of diphtheria toxin protein A (dt-a) to selectively induce cell death of DA neurons. Here we use Cre also to activate expression of the fluorescent protein mCherry from AAV-LSL-mCherry.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4546329&req=5

pone.0136203.g006: Schematic overview of utilized transgenic mice and recombinant AAV (rAAV) constructs.TH-tTA and TH-rtTA mice express, under the tyrosine hydroxylase (TH) promoter, the tetracycline-regulated transactivator protein (tTA) or the reverse tetracycline-regulated transactivator protein (rtTA) in dopaminergic (DA) neurons. tTA binds to the tetracycline operon (tetO) in the absence of doxycycline (DOX) and rtTA binds in the presence of DOX, driving transient expression of gene X, for example the fluorescent protein Venus from a recombinant AAV vector (AAV-tetO-Venus), or luciferase and Cre from the LC1 construct. The LC1 encodes a luciferase enzyme, which can be used for bioluminescence in vivo imaging, and the Cre recombinase for deleting gene Y or removing floxed STOP codons (LSL). Cre is used here as a genetic switch to activate, from the ROSA locus, either expression of the reporter gene lacZ (encoding β-galactosidase) to visualize transgene expression or to activate expression of diphtheria toxin protein A (dt-a) to selectively induce cell death of DA neurons. Here we use Cre also to activate expression of the fluorescent protein mCherry from AAV-LSL-mCherry.
Mentions: LC1 mice [21], Rosa26R reporter mice [22] and ROSA26-dt-a mice [23] were described previously (Figs 1–6).

Bottom Line: The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson's disease (PD) and drug addiction.Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification.We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre.

View Article: PubMed Central - PubMed

Affiliation: Development and Maintenance of the Nervous System, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT

Background & aims: The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson's disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification.

Methods & results: Here, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre. In combination with mice encoding tet-responsive luciferase, we visualized the DA system in living mice progressively over time.

Conclusion: These experiments establish TH-tTA and TH-rtTA mice as a powerful tool to generate and monitor mouse models for DA system diseases.

No MeSH data available.


Related in: MedlinePlus