4 research outputs found
Cre independent adult gene expression in DA neurons of TH-tTA and TH-rtTA mice using a AAV-tetO-Venus vector.
<p><b>(A)</b> Scheme of fluorescent DA neuron labelling in non-DOX treated TH-tTA mice stereotactically injected with AAV-tetO-Venus vector in the ventral midbrain. <b>(B)</b> Confocal fluorescent pictures of Venus expression (green) in DA neurons co-stained for tyrosine hydroxylase (TH; red) in the substantia nigra pars compacta (SNpc) of sagittal TH-tTA mouse brain sections. <b>(C)</b> Scheme of fluorescent DA neuron labeling in DOX-treated TH-rtTA mice stereotactically injected with AAV-tetO-Venus vector in the ventral midbrain. <b>(C)</b> Scheme of fluorescent DA neuron labelling in DOX-treated TH-rtTA mice stereotactically injected with AAV-tetO-Venus vector in the ventral midbrain. <b>(D)</b> Confocal fluorescent pictures of Venus expression in DA neurons co-stained for TH in the substantia nigra pars compacta (SNpc) of sagittal TH-rtTA mouse brain sections. <b>(E)</b> Quantification reveals that in the ON-state 67% and 73% and in the OFF-state 27% and 23% of TH+ cells are also Venus-positive in TH-tTA and TH-rtTA mice, respectively. In non-transgenic mice (controls) only 5% of TH+ cells were also Venus-positive. n = 3–5. Scale bars: 250 μm and in high magnification picture 50 μm.</p
Efficient fluorescent labeling of DA neurons and their axons in AAV-LSL-mCherry injected DA neuron-specific Cre mice.
<p><b>(A)</b> Scheme for specific fluorescent labeling of DA neuron in mice expressing a DA neuron-specific Cre and stereotaxic injected in the mindbrain with a recombinant AAV vector expressing mCherry after a floxed STOP codon. <b>(B-G)</b> Fluorescent mCherry expression in DA cells of the midbrain (B-D) and DA fibers in the striatum (E-G) in sagittal sections of stereotaxic injected DAT-Cre mice. Expression was evaluated 1 (B,E), 3 (C,F) and 5 weeks (D,G) after injection. After 1 week, no expression could be detected in DA fibers of the striatum. Expression in DA fibers was saturated 5 weeks after injection. Scale bars = 200 μm (B-D); 20 μm (E-G). <b>(H-O)</b> Fluorescent mCherry expression in DA cells of the midbrain (H-K) and DA fibers in the striatum (L-O) after stereotaxic injection of TH-tTA/LC1 mice. Fluorescent expression was evaluated at the indicated time points in mice raised without DOX. Some mice were kept on DOX-containing food after the virus injection until analyzed (J, K, N, O). Scale bars = 200 μm (H-K); 20 μm (L-O). <b>(P and Q)</b> No fluorescent mCherry expression was detected in the lateral habenula (LHb) of AAV-LSL-mCherry injected DAT-Cre (P) and TH-tTA/LC1 (Q) mice after 5 weeks. Scale bars = 200 μm.</p
Specificity, inducibility and efficacy of gene expression in TH-tTA/LC1/Rosa26R and TH-rtTA/LC1/Rosa26R mice.
<p><b>(A)</b> Genetic scheme of DOX-regulated reporter gene expression in TH-tTA and TH-rtTA mice crossed with LC1 and Rosa26R reporter mice. <b>(B and C)</b> Coronal midbrain brain sections with substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) DA neurons of TH-tTA/LC1/Rosa26R (B) and TH-rtTA/LC1/Rosa26R (C) mice raised with or without doxycycline (DOX) or transiently with DOX to have the system switched off during development (TH-tTA mice treated with DOX during pre- and postnatal development till 6 weeks of age; TH-rtTA mice raised without DOX) and on during analysis (TH-tTA mice from the age of 6 weeks without DOX; TH-rtTA mice from the age of 6 weeks with DOX). Sections were stained for β-galactosidase activity with X-gal to visualize cells with activated tet-system. Adjacent sections were X-gal stained and co-stained with tyrosine hydroxylase (TH) antibodies and DAB substrate (brown) to mark DA neurons. <b>(D)</b> Zoom in picture of TH and lacZ double positive cells in the SNpc. <b>(E and F)</b> Quantification of TH and lacZ double positive cells (LacZ+TH+) in the SNpc and VTA to estimate recombination efficacy in DA neurons (TH+) of animals treated constantly (constant DOX), transiently (transient DOX) or without DOX (no DOX) in the tet-OFF (E) and tet-ON mice (F). mean + s.e.m.; n = 3. Scale bars: 500 μm (B and C), 50 μm (D).</p
Schematic overview of utilized transgenic mice and recombinant AAV (rAAV) constructs.
<p>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 <i>in vivo</i> 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.</p