TTA / TetOff Atlas (PrP)

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Intro-image PrP.jpg

PrPpromoter is an online public neuroscience data repository based on data published in Boy et al, NeuroImage 2006; 33:449-462. It provides access to a collection of high resolution brain section images from a double transgenic mouse line (Prnp-tTA:βGal), obtained by crossbreeding a tetracycline-responsive prion protein promoter mouse line (Tremblay et al., 1998) with a responder mouse line transgenic for a reporter gene construct containing the LacZ reporter gene (encoding the enzyme β-galactosidase). The images can be viewed with a Virtual Microscope viewing tool that allows inspection of the PrP promoter distribution at atlas coordinates and magnifications chosen by the user. Each section has been annotated with names of key landmarks, regions, nuclei, and areas, in accordance with mouse brain stereotaxic atlas of Paxinos and Franklin (2001).

On this page you can learn more about this application, access the image repository, and navigate across and within the section images. For an exemplification of content, go to the Demo section.

PrPpromoter is part of the Rodent Brain WorkBench, a new research and development project funded by The Research Council of Norway, the Centre for Molecular Biology and Neuroscience, and the European Union. The research that has produced the present material has been supported by Fritz Thyssen Stiftung, the Deutsche Heredo-Ataxie-Gesellschaft, and the European Union (Marie Curie EST programme, HPMT-CT-2001-00406-03).


Reference

Boy J, Leergaard TB, Schmidt T, Odeh F, Bichelmeier U, Nuber S, Holzmann C, Wree A, Prusiner SB, Bujard HB, Riess O, Bjaalie JG
Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease-models. 
NeuroImage 2006; 33:449-462


Examples

Bregma 1.73 mm

Bregma -0.22 mm

Bregma -1.42 mm

Bregma -2.82 mm

Bregma -5.02 mm


Access repository and Virtual Microscope

The present Virtual Microscope viewing tool allows the user to navigate within and across 60 coronal section images from the brain of a tetracycline-responsive prion protein (PrP) promoter mouse. It allows inspection of the PrP promoter activity at defined atlas levels and at chosen magnification throughout the brain. Each section image has been annotated with names of key landmarks, regions, nuclei, and areas.

Re-use of data from this repository is allowed provided that reference is given to the following publication:

Boy J, Leergaard TB, Schmidt T, Odeh F, Bichelmeier U, Nuber S, Holzmann C, Wree A, Prusiner SB, Bujard HB, Riess O, Bjaalie JG
Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease-models. 
NeuroImage 2006; 33:449-462


Block name:       Block Date:        Cutting direction:

PrPBrain              2005-11-09          Coronal

 

Experimental procedures

Overview

1.    Double transgenic (Prnp-tTA:βGal) mice were produced by crossbreeding a tetracycline responsive PrP promoter mouse lines with responder mice transgenic for the LacZ reporter gene (encoding the enzyme β-galactosidase). 

2.   Isolated paraformaldehyde-fixed brains were coronally sectioned at 25 µm using a cryostat. 

a.    Cells expressing the PrP promoter produce β-galactosidase and are labeled blue using X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) as a substrate.

b.    Weak counterstaining with Neutral Red facilitated detection of boundaries and landmarks, without compromising the observation of X-Gal labeling or interfering with the labeling pattern. 

3.   High-resolution mosaic images were obtained from coronal sections with use of robotic microscopy.

 

Gene construct

The tetracycline-responsive (TetR) gene system is a binary transgenic system in which in which transgene expression may be activated or silenced by administration of tetracycline, or its derivates (Gossen and Bujard, 1992; Gossen et al., 1995). Such animal models are generated by crossing two different mouse lines, one with a gene modulator (promoter mouse line) and one with the actual disease-causing gene (responder mouse line). In the double transgenic offspring both the regional distribution and intensity of target gene expression depend on the activity of the chosen promoter controlling the expression of a gene activator, which in turn is accessible for experimental modulation.

The double transgenic mice used here were produced by crossbreeding a Prnp-tTA mouse line containing the hamster PrP promoter gene (Tremblay et al., 1998) with a responder mouse line transgenic for a bidirectional reporter gene construct containing both the Luciferase and LacZ reporter gene (Baron et al., 1995; Schönig and Bujard, 2003), which encodes the enzyme β-galactosidase. The resulting double transgenic mice are referred to as Prnp-tTA:βGal, in which inducible LacZgene expression is under control of the PrP gene (Prnp) promoter. The PrP promoter was obtained from hamster, theLuciferase gene (which was not employed in this study) from Photinus pyralis, and the LacZ gene from E. coli. The LacZgene product (β-galactosidase) was identified using X-gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside) as a substrate.

Gene-construct prp.jpg
The tetracycline-dependent regulatory (Tet-Off) system. The Prnp promoter construct controls the expression of the tetracycline transactivator (tTA) gene product, which induces the transcription of the responder construct by binding to a tetracycline responsive element (TRE). The gene expression can be blocked by tetracycline (Tc). For simplification, the second part of the bidirectional construct containing theLuciferase gene is omitted. Drawing modified after a scheme by BD Biosciences Clontech (BDTM Tet-On and Tet-Off Systems brochure; www.bdbiosciences.com). Abbreviations: AD, VP16 activation domain of herpes simplex virus; LacZ,LacZ gene encoding β-galactosidase; PminCMV, minimal CMV promoter; Prnp, PrP gene; Tc, Tetracycline; TetR, Tet repressor protein; tTA, tetracycline transactivator; TRE, tetracycline responsive element.

Western blot

Regionally specific promoter distribution revealed by Western blot analysis of separate brain regions

Histology

In the double transgenic Prnp-tTA:βGal mice, PrP promoter activity regulates the expression of the LacZ reporter gene which encodes for the enzyme β-galactosidase (Baron et al., 1995, Schönig and Bujard, 2003). Cells that contain this enzyme are identified using X-gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside) as a β-galactosidase substrate. Enzymatic cleavage of X-gal gives rise to an insoluble indigo-blue compound (Holt and O’Sullivan, 1958; Cepko et al., 1998), which is visible both at the macroscopic level in whole brains, and microscopically in histological sections (see Figure below)


Overview of the histological procedures

Following deep anaesthesia, mice were transcardially perfused with 4% paraformaldehyde. Dissected whole brains were sectioned at 25 µm on a cryostat. Every eight consecutive section (200 µm spacing) was collected for processing as free-floating sections with X-gal. Stained sections were mounted on glass slides, counterstained with Neutral Red, and coverslipped.

To eliminate the possibility of endogenous β-galactosidase activity contributing to the labeling pattern, brain sections from control animals (the LacZ responder line and wild-type C57Bl/6 mice) were stained with X-gal.

Histology prp.jpg
Examples illustrating the specificity of X-gal labeling. Images from coronal sections at the level of the substantia nigra in brains from control animals (a: LacZ and c: C57BL/6) and Prnp-tTA:βGal double transgenic animals (b, d), incubated with X-gal. X-gal labeled cells, indicating PrP promoter expression, are only found in the brains of Prnp-tTA:βGal animals. Counterstaining of sections with Neutral Red (c,d) facilitates delineation of anatomical landmarks and architectural features without compromising the X-gal labeling. Abbreviations: cp, cerebral peduncle; SNR, substantia nigra pars reticularis. Bar, 200 µm.



To facilitate localization and comparison of brain elements with stereotaxic brain atlases, detailed photographic documentation of the whole brain (A and B) was performed before sectioning. Special care was also taken to define section angle and to monitor section position relative to the intact dissected brain (C, device for defining section angle). Figure reproduced from Bjaalie and Leergaard (2005).
Blocking-device histology prp.jpg

Protocols

Transcardial Perfusion

a.      Surgical anaesthesia was obtained by intraperitoneal injection (0,075 ml per 10g, ~ 0,3 ml) of a mixture of equal volumes of Hypnorm (Janssen Pharmaceutical, Beerse, Belgium) and Dormicum (5 mg/ml; F. Hoffmann – La Roche, Basel, Switzerland).

b.     The animals were transcardially perfused with lukewarm phosphate-buffered saline, followed by 35 ml of freshly made, lukewarm 4% paraformaldehyde in phosphate-buffered saline (pH 7,4) and finally cold 10% sucrose in 0,1 M NaPi (phosphate buffer 0,08 M Na2HPO4; 0,02 M NaH2PO4).

c.      The brains were removed and stored overnight at 4°C in 30% sucrose (in 0,1 M NaPi).


4% paraformaldehyde in phosphate-buffered saline (pH 7,4)

Use 4 g paraformaldehyde (PFA) per 100 ml phosphate-buffered saline (PBS). Mix 4 g PFA with less than 100 ml PBS (pH 7,4) and heat solution (to ~70°C, under hood) while stirring. Dissolve PFA by making the solution alkaline using a few drops of 1M NaOH. Let the solution cool to room temperature and adjust pH to 7,4 by adding HCl and fill up to 100 ml.


Gelatin embedding and cryoprotection

a.      Rinse the brain thoroughly in PBS, remove meninges if present.

b.     Submerge whole brain in 5% gelatin (in 0,1 M NaPi) and keep at 37°C for 1-2 hours.

c.      Change to 7% gelatin solution and keep at 37°C for another 1-2 hours.

d.      Make a horizontal gelatin platform in a peel-a-way mould (~2 mm thick layer of fresh gelatin, harden in freezer).

e.      Position the brain on the gelatin platform at square angles in the mould such that the longitudinal cerebral fissure (midline) is parallel to the sides of the mould, and the baseline of the brain is parallel to the bottom of the mould (see also “defining the section angle” below).

f.      Embed the brain in fresh 7% gelatin and keep in freezer for approximately 10-20 minutes until the gelatin hardens.

g.      Remove the peel-a-way mould and denaturate the gelatin block by immersion in 4% PA for 4 hours at 4°C.

h.     Immerse overnight at 4°C in 30% sucrose for cryoprotection.


Defining the section angle

a.      Adjust the surface underlying the brain such that the cortex approximately underlying bregma and the apex of cerebellum are at the same horizontal level (C in figure above), and that the midline of the brain is perpendicular to the cutting blade.

b.     Trim away excess gelatin. To keep track of left and right positions of the sections, the gelatin block is made asymmetric by applying a wedge cut into the dorsal right side of the block.


X-gal staining of free-floating sections

a.      10 min permeabilization in 0,01 deoxycholic acid + 0,02 % NP-40 (Igepal) in PBS at room temperature.

b.     10 min 1x PBS at roomtemp.

c.      3-4 hours incubation in X-gal staining solution (at 30°C, keep dark): 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2mM MgCl2, 250 µg/ml X-gal in PBS pH 7,4.

d.      Stop staining by washing sections with PBS (1-2 times).

e.      Mount sections on to gelatin coated glass slides (or SuperFrost glasses) with NaPi 0,1 M and dry overnight.

f.      Cover sections with Eukitt (dried sections directly into xylene 2x).


Solutions

K3Fe(CN)6 - 200 mM in H2O, protected from light, stored at 4°C
K4Fe(CN)6 - 200 mM in H2O, protected from light, stored at 4°C (made fresh).
MgCl2 - 200 mM in H2O store at room temperature.
X-gal - 10 – 20 mg/ml dissolved in DMSO in a glass vessel, protected from light, store at -20°C, thaw before use.


Neutral Red Counterstaining of X-gal stained sections
  • 1 % Neutral Red
  • Sections on glass slides are directly dipped into Neutral Red for ~5 min
  • 2x wash in 70% ethanol (each 10 sec)
  • 90% ethanol
  • 2x 100% ethanol (very short)
  • 2x xylene
  • Sections are cover slipped with Eukitt

 

Image acquisition

High-resolution mosaic images of the sections were obtained through an automated Olympus Bx52 microscope, equipped with a high-precision motorized stage (LEP MAC5000, LUDL Electronic Products Ltd., Hawthorne, NY, USA), an Optronics MicroFire digital camera (Optronics Picture This, Goleta, CA USA), and Neurolucida v6.0 Virtual Slice software (MicroBrightField Inc., Williston, VT, USA). With use of this system, mosaic images (consisting of around 150 individual frames) were obtained from entire coronal brain sections, using a 20x lens. The resulting images (tiff file format; 300-400 MB) provide a nearly seamless overview of entire sections, with high resolution to distinguish individual cell morphology.


Olympus Bx52 microscope, equipped with a high-precision motorized MAC5000 LUDL stage and an Optronics MicroFire digital camera.
Individual frame from the cerebellar cortex, captured with a 20x lens. Gr: Granular layer, Mol: Molecular layer, Py: Purkinje cell layer. Scale bar: 200 µm
Coronal section ~ 9 x 5 mm 25 micrometer section thickness. Data acquisition: 20x lens, digital camera, high precision motorized stage, software controlled frame-by-frame acquisition. Composite image of 136 individual frames per section, ~ 400 Mb tiff file, ~ 5 min’s acquisition time

 

Ontologies and coordinate system

All coordinates, annotations and abbreviations used to label and facilitate section identification, are in accordance with the mouse brain atlas by G. Paxinos and K.B.J. Franklin: The Mouse Brain in Stereotaxic Coordinates, Academic Press, 2001.

In certain regions, we identified the region or a nuclear group as a whole, and not sub-regions or individual nuclei. This necessitated the adoption of abbreviations not present in the Paxinos and Franklin atlas, e.g. “AA” used to label the Amygdaloid Area. For clarification, each section image is accompanied with a list of abbreviations used to annotate the image, with their meaning.

The standard global, skull based coordinate system, as described in the Paxinos and Franklin atlas, was used for assigning coordinates to the individual sections, by comparing the experimental sections one by one with the atlas sections, and by taking into consideration the order and spacing between the sections and distances to known landmarks. Due to variation in brain size and dimensions of regions between individual animals, we provide two sets of coordinates for each section. The first is our estimate of the Bregma value in a given section, the second is the “Atlas Bregma” value (together with the corresponding “Interaural” value) for the Paxinos and Franklin atlas section closest to a given experimental section


Contributing laboratories

Department of Medical Genetics - Jana Boy, Thorsten Schmidt, Ulrike Bichelmeier, Silke Nuber, Olaf Riess
University of Tübingen
Calwerstrasse 7
D-72076 Tübingen
Germany

Department of Medical Genetics - Carsten Holzmann
University of Rostock
Rembrandtstrasse 16/17
D-18057 Rostock
Germany

Department of Anatomy - Andreas Wree
University of Rostock
Gertrudenstrasse 9
D - 18057 Rostock
Germany

Institute for Neurodegenerative Diseases - Stanley Prusiner
University of California
Box 0518
San Francisco
CA 94143-0518
USA

ZMBH (Zentrum für Molekulare Biologie Heidelberg) – Hermann Bujard
University of Heidelberg
Im Neuenheimer Feld 282
D-69120 Heidelberg
Germany

Neural Systems and Graphics Computing Laboratory - Trygve B. Leergaard, Francis Odeh, Jan G. Bjaalie
Centre for Molecular Biology and Neuroscience &
Institute of Basic Medical Sciences - Anatomy
University of Oslo
P.O. Box 1105 Blindern
N - 0317 Oslo
Norway
http://www.nesys.uio.no  

http://www.cmbn.no

 

Credits

PrPpromoter web-application development team:
Jan Olav Kjøde
Ivar Andre Moene 
Francis Odeh
Trygve B. Leergaard
Jan G. Bjaalie

References

Boy J, Leergaard TB, Schmidt T, Odeh F, Bichelmeier U, Nuber S, Holzmann C, Wree A, Prusiner SB, Bujard HB, Riess O, Bjaalie JG
Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease-models. 
NeuroImage 2006; 33:449-462

Baron U, Freundlieb S, Gossen M, Bujard H
Co-regulation of two gene activities by tetracycline via a bidirectional promoter.
Nucleic Acids Res. 1995; 23:3605-3606

Bjaalie JG
Localization in the brain: new solutions emerging.
Nature Neurosci Reviews 2002; 3:322-325

Bjaalie JG, Leergaard TB
Three-dimensional computerized reconstruction from serial sections: cells populations, regions, and whole brain.
In Neuroanatomical Tract Tracing: Molecules, Neurons, & Systems (eds. Zaborszky,L., Wouterlood,F.G. & Lanciego,J.L.) 2005; In Press (Springer Science+Business Media, New York)

Cepko C, Ryder E, Fekete DM, Bruhn S
Detection of beta-galactosidase and alkaline phosphatase activities in tissue.
In Cells: A Laboratory Manual, Volume 3: Subcellular Location of Genes and Their Products (eds. Spector,D.L., Goldman,R.D. & Leinwand,L.A.) 1998; Cold Spring Harbor Laboratory Press, Cold Spring Harbor

Gossen M, Bujard H
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
Proc Natl Acad Sci USA 1992; 89:5547-5551

Gossen M, et al.
Transcriptional activation by tetracyclines in mammalian cells.
Science 1995; 268:1766-1769

Holt S, O'Sullivan D
Studies in enzyme cytochemistry I. Principles of cytochemical staining methods.
Proc R Soc Lond B Biol Sci. 1958; 148:465-480

Paxinos G, Franklin KBJ
The Mouse Brain in Stereotaxic Coordinates
Academic Press, San Diego 2001

Schönig K, Bujard H
Generating conditional mouse mutants via tetracycline-controlled gene expression.
In Transgenic Mouse Methods and Protocols (eds. Hofker,M. & van Deursen,J.) Humana Press, Totowa, NJ. 2003; 69-104

Tremblay P, et al.
Doxycycline control of prion protein transgene expression modulates prion disease in mice.
Proc Natl Acad Sci USA 1998; 95:12580-12585