Difference between revisions of "TTA / TetOff Atlas (PrP)"

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[[File:Intro-image PrP.jpg|thumb|right|link=#Examples]]
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== About ==
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The tetracycline-responsive (TetR) gene system is a binary transgenic system in which transgene expression may be activated or silenced by administration of tetracycline, or its derivates (Gossen and Bujard, PNAS 89:5547-5551, 1992; Gossen et al., Science 286:1766-1769, 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. Detailed knowledge about where in the brain gene expression is regulated is essential for the use and interpretation of such models.
  
''PrPpromoter''<span style="font-family: georgia, serif;">&nbsp;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).</span>
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The TetOff atlas 1 (PrPpromoter) is an interactive resource providing access to a collection of microscopic brain section from a double transgenic mouse line (Prnp-tTA:βGal), obtained by crossbreeding a tetracycline-responsive prion protein promoter mouse line with a responder mouse line transgenic for a reporter gene construct containing the LacZ reporter gene (encoding the enzyme β-galactosidase).
  
<span style="font-family:georgia,serif;">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.</span>
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== <span style="font-family:georgia,serif">Access image repository</span> ==
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The virtual microscopy viewer allows interactive zooming and panning. Original images are available for download via separate link. Each section has been annotated with names of key landmarks, regions, nuclei, and areas.
  
<span style="font-family:georgia,serif;">''PrPpromoter''&nbsp;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).</span>
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'''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 33:449-462, 2006. 
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{| class="wikitable"
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!Animal #
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!Genotype
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!Image repository
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!Download original
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|-
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|388.12
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|prp-lacZ
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|[http://cmbn-navigator.uio.no/navigator/filmstrip_viewer.html?publicOnly=true&entityType=block&entityId=2541&selectIndex=17&metadataKeyName=Bregma%20Level# Filmstrip viewer]
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|[http://cmbn-navigator.uio.no/navigator/feeder/all_originals/?id=2541 Tiffs]
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|}
  
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== <span style="font-family:georgia,serif">Experimental procedures in brief</span> ==
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<span style="font-family:georgia,serif"><div style="clear:both">Double transgenic mice were produced by crossbreeding a Prnp-tTA mouse line containing the hamster PrP promoter gene (Tremblay et al., PNAS 95:12580-12585,1998) with a responder mouse line transgenic for a bidirectional reporter gene construct containing both the Luciferase and LacZ reporter gene (Baron et al., Nucleic Acids Res. 23:3605-3606, 1995), which encodes the enzyme β-galactosidase. In the resulting double transgenic mice (Prnp-tTA:βGal) 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.
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Anaesthetized mice were transcardially perfused with 4% paraformaldehyde. Dissected whole brains were sectioned at 25 µm on a cryostat. Every eighth 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.
  
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High-resolution mosaic section images 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).
  
== <span style="font-family:georgia,serif;">Reference</span> ==
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All coordinates, annotations and abbreviations used to label and facilitate section identification, are in accordance with the mouse brain atlas by Paxinos and Franklin: The Mouse Brain in Stereotaxic Coordinates, Academic Press, 2001.
  
<span style="font-family:georgia,serif;">'''Boy J, Leergaard TB, Schmidt T, Odeh F, Bichelmeier U, Nuber S, Holzmann C, Wree A, Prusiner SB, Bujard HB, Riess O, Bjaalie JG'''<br/>''Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease-models.&nbsp;''<br/>NeuroImage 2006; 33:449-462</span>
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For further details, see Boy et al. (NeuroImage, 33:449-462, 2006).
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</div>
  
 
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== Contributing laboratories ==
 
 
== <span style="font-family:georgia,serif;">Examples</span> ==
 
 
 
<span style="font-family:georgia,serif;">'''1. Bregma 1.73 mm'''</span>
 
 
 
<gallery>
 
File:Section1a.jpg|1
 
File:Section1b.jpg|2
 
File:Section1c.jpg|3
 
File:Section1.gif|Overview
 
</gallery>
 
 
 
<span style="font-family:georgia,serif;">'''2. Bregma -0.22 mm'''</span>
 
 
 
<gallery>
 
File:Section2a.jpg|1
 
File:Section2b.jpg|2
 
File:Section2c.jpg|3
 
File:Section2.gif|Overview
 
</gallery>
 
 
 
<span style="font-family:georgia,serif;">'''3. Bregma -1.42 mm'''</span>
 
 
 
<gallery>
 
File:Section3a.jpg|1
 
File:Section3b.jpg|2
 
File:Section3c.jpg|3
 
File:Section3.gif|Overview
 
</gallery>
 
 
 
<span style="font-family:georgia,serif;">'''4. Bregma -2.82 mm'''</span>
 
 
 
<gallery>
 
File:Section4a.jpg|1
 
File:Section4b.jpg|2
 
File:Section4c.jpg|3
 
File:Section4.gif|Overview
 
</gallery>
 
 
 
<span style="font-family:georgia,serif;">'''5. Bregma -5.02 mm'''</span>
 
 
 
<gallery>
 
File:Section5a.jpg|1
 
File:Section5b.jpg|2
 
File:Section5c.jpg|3
 
File:Section5.gif|Overview
 
</gallery>
 
 
 
== <span style="font-family:georgia,serif;">Access image repository</span> ==
 
 
 
<span style="font-family:georgia,serif;">The present 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.</span>
 
 
 
<span style="font-family:georgia,serif;">Re-use of data from this repository is allowed provided that reference is given to the following publication:</span>
 
 
 
<span style="font-family:georgia,serif;">'''Boy J, Leergaard TB, Schmidt T, Odeh F, Bichelmeier U, Nuber S, Holzmann C, Wree A, Prusiner SB, Bujard HB, Riess O, Bjaalie JG'''<br/>''Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease-models.&nbsp;''<br/>NeuroImage 2006; 33:449-462</span>
 
 
 
 
 
 
 
<span style="font-family:georgia,serif;">'''Block name:&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Block Date:&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Cutting direction:'''</span>
 
 
 
<span style="font-family:georgia,serif;">[http://cmbn-approd01.uio.no/zoomgen/zoomify_viewer.do?initial=695&width=1920&height=1080 PrPBrain] &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;2005-11-09 &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Coronal</span>
 
 
 
<span style="font-family:georgia,serif;">&nbsp;</span>
 
 
 
== <span style="font-family:georgia,serif;">Experimental procedures</span> ==
 
 
 
=== <span style="font-family:georgia,serif;">Overview</span> ===
 
 
 
<span style="font-family:georgia,serif;">1.&nbsp;&nbsp;&nbsp; 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).&nbsp;</span>
 
 
 
<span style="font-family:georgia,serif;">2.&nbsp;&nbsp; Isolated paraformaldehyde-fixed brains were coronally sectioned at 25 µm using a cryostat.&nbsp;</span>
 
 
 
<span style="font-family:georgia,serif;">a.&nbsp;&nbsp;&nbsp; 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.</span>
 
 
 
<span style="font-family:georgia,serif;">b.&nbsp;&nbsp;&nbsp; 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.&nbsp;</span>
 
 
 
<span style="font-family:georgia,serif;">3.&nbsp;&nbsp; High-resolution mosaic images were obtained from coronal sections with use of robotic microscopy.</span>
 
 
 
<span style="font-family:georgia,serif;">&nbsp;</span>
 
 
 
=== <span style="font-family:georgia,serif;">Gene construct</span> ===
 
 
 
<span style="font-family:georgia,serif;">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.</span>
 
 
 
<span style="font-family:georgia,serif;">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&nbsp;''Luciferase''&nbsp;and&nbsp;''LacZ''&nbsp;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&nbsp;''LacZ''gene expression is under control of the PrP gene (Prnp) promoter. The PrP promoter was obtained from hamster, the''Luciferase''&nbsp;gene (which was not employed in this study) from&nbsp;''Photinus pyralis'', and the&nbsp;''LacZ''&nbsp;gene from&nbsp;''E. coli''. The&nbsp;''LacZ''gene product (β-galactosidase) was identified using X-gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside) as a substrate.</span>
 
 
 
<span style="font-family:georgia,serif;">[[File:Gene-construct prp.jpg|frame|left]]The tetracycline-dependent regulatory (Tet-Off) system. The&nbsp;''Prnp''&nbsp;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 the''Luciferase''&nbsp;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;&nbsp;''LacZ'',''LacZ''&nbsp;gene encoding β-galactosidase; P<sub>minCMV</sub>, minimal CMV promoter;&nbsp;''Prnp'', PrP gene; Tc, Tetracycline; TetR, Tet repressor protein; tTA, tetracycline transactivator; TRE, tetracycline responsive element.</span>
 
 
<div style="clear:both">
 
<div style="clear:both">
=== <span style="font-family:georgia,serif;">Western blot</span> ===
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* '''Department of Medical Genetics''', University of Tübingen, Calwerstrasse 7, D-72076 Tübingen, Germany. '''People''': Jana Boy, Thorsten Schmidt, Ulrike Bichelmeier, Silke Nuber, Olaf Riess
 
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* '''Department of Medical Genetics''', University of Rostock, Rembrandtstrasse 16/17, D-18057 Rostock, Germany. '''People''': Carsten Holzmann
<span style="font-family:georgia,serif;">[[File:Westernblot prp.jpg|Regionally specific promoter distribution revealed by Western blot analysis of separate brain regions]]</span>
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*  '''Department of Anatomy''', University of Rostock, Gertrudenstrasse 9, D - 18057 Rostock, Germany. '''People''': Andreas Wree
 
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*  '''Institute for Neurodegenerative Diseases''', University of California, San Franscisco, Box 0518 San Francisco, CA 94143-0518, USA. '''People''': Stanley Prusiner
=== <span style="font-family:georgia,serif;">Histology</span> ===
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*  '''ZMBH (Zentrum für Molekulare Biologie Heidelberg)''', University of Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany. '''People''': Hermann Bujard
 
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*  '''Neural Systems Laboratory''' (http://www.nesys.uio.no), Centre for Molecular Biology and Neuroscience & Institute of Basic Medical Sciences, Department of Anatomy, University of Oslo, P.O. Box 1105 Blindern, N - 0317 Oslo, Norway: Histological processing, image acquisition, atlas repository. '''People''': Francis Odeh, Jan O. Kjøde, Ivar A. Moene, Trygve B. Leergaard, Jan G. Bjaalie
<span style="font-family:georgia,serif;">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)</span>
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</div>
 
 
 
 
 
 
==== <span style="font-family:georgia,serif;">Overview of the histological procedures</span> ====
 
 
 
<span style="font-family:georgia,serif;">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&nbsp;[http://en.wikipedia.org/wiki/X-gal X-gal]. Stained sections were mounted on glass slides, counterstained with&nbsp;[http://en.wikipedia.org/wiki/Neutral_red Neutral Red], and coverslipped.</span>
 
 
 
<span style="font-family:georgia,serif;">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.</span>
 
 
 
<span style="font-family:georgia,serif;">[[File:Histology prp.jpg|frame|left|50px]]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.</span>
 
 
 
<br/>[[File:Blocking-device histology prp.jpg|frame|center|50px]]<br/><span style="font-family:georgia,serif;">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).</span>
 
<div style="clear:both"></div><br/><br/>
 
 
 
==== <span style="font-family:georgia,serif;">Protocols</span> ====
 
 
 
===== <span style="font-family:georgia,serif;">Transcardial Perfusion</span> =====
 
 
 
<span style="font-family:georgia,serif;">a.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 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).</span>
 
 
 
<span style="font-family:georgia,serif;">b.&nbsp;&nbsp;&nbsp;&nbsp; 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).</span>
 
 
 
<span style="font-family:georgia,serif;">c.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; The brains were removed and stored overnight at 4°C in 30% sucrose (in 0,1 M NaPi).</span>
 
 
 
 
 
 
 
===== <span style="font-family:georgia,serif;">4% paraformaldehyde in phosphate-buffered saline (pH 7,4)</span> =====
 
 
 
<span style="font-family:georgia,serif;">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.</span>
 
 
 
 
 
 
 
===== <span style="font-family:georgia,serif;">Gelatin embedding and cryoprotection</span> =====
 
 
 
<span style="font-family:georgia,serif;">a.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Rinse the brain thoroughly in PBS, remove meninges if present.</span>
 
 
 
<span style="font-family:georgia,serif;">b.&nbsp;&nbsp;&nbsp;&nbsp; Submerge whole brain in 5% gelatin (in 0,1 M NaPi) and keep at 37°C for 1-2 hours.</span>
 
 
 
<span style="font-family:georgia,serif;">c.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Change to 7% gelatin solution and keep at 37°C for another 1-2 hours.</span>
 
 
 
<span style="font-family:georgia,serif;">d.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Make a horizontal gelatin platform in a peel-a-way mould (~2 mm thick layer of fresh gelatin, harden in freezer).</span>
 
 
 
<span style="font-family:georgia,serif;">e.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 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).</span>
 
 
 
<span style="font-family:georgia,serif;">f.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Embed the brain in fresh 7% gelatin and keep in freezer for approximately 10-20 minutes until the gelatin hardens.</span>
 
 
 
<span style="font-family:georgia,serif;">g.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Remove the peel-a-way mould and denaturate the gelatin block by immersion in 4% PA for 4 hours at 4°C.</span>
 
 
 
<span style="font-family:georgia,serif;">h.&nbsp;&nbsp;&nbsp;&nbsp; Immerse overnight at 4°C in 30% sucrose for cryoprotection.</span>
 
 
 
 
 
 
 
===== <span style="font-family:georgia,serif;">Defining the section angle</span> =====
 
 
 
<span style="font-family:georgia,serif;">a.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 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.</span>
 
 
 
<span style="font-family:georgia,serif;">b.&nbsp;&nbsp;&nbsp;&nbsp; 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.</span>
 
 
 
 
 
 
 
===== <span style="font-family:georgia,serif;">X-gal staining of free-floating sections</span> =====
 
 
 
<span style="font-family:georgia,serif;">a.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 10 min permeabilization in 0,01 deoxycholic acid + 0,02&nbsp;% NP-40 (Igepal) in PBS at room temperature.</span>
 
 
 
<span style="font-family:georgia,serif;">b.&nbsp;&nbsp;&nbsp;&nbsp; 10 min 1x PBS at roomtemp.</span>
 
 
 
<span style="font-family:georgia,serif;">c.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3-4 hours incubation in X-gal staining solution (at 30°C,&nbsp;keep dark): 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2mM MgCl2, 250 µg/ml X-gal in PBS pH 7,4.</span>
 
 
 
<span style="font-family:georgia,serif;">d.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Stop staining by washing sections with PBS (1-2 times).</span>
 
 
 
<span style="font-family:georgia,serif;">e.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Mount sections on to gelatin coated glass slides (or SuperFrost glasses) with NaPi 0,1 M and dry overnight.</span>
 
 
 
<span style="font-family:georgia,serif;">f.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Cover sections with Eukitt (dried sections directly into xylene 2x).</span>
 
 
 
 
 
 
 
===== <span style="font-family:georgia,serif;">Solutions</span> =====
 
 
 
<span style="font-family:georgia,serif;">K3Fe(CN)6 - 200 mM in H2O, protected from light, stored at 4°C<br/>K4Fe(CN)6 - 200 mM in H2O, protected from light, stored at 4°C (made fresh).<br/>MgCl2 - 200 mM in H2O store at room temperature.<br/>X-gal - 10 – 20 mg/ml dissolved in DMSO in a glass vessel, protected from light, store at -20°C, thaw before use.</span>
 
 
 
 
 
 
 
===== <span style="font-family:georgia,serif;">Neutral Red Counterstaining of X-gal stained sections</span> =====
 
 
 
*<span style="font-family:georgia,serif;">1&nbsp;% Neutral Red</span>
 
*<span style="font-family:georgia,serif;">Sections on glass slides are directly dipped into Neutral Red for ~5 min</span>
 
*<span style="font-family:georgia,serif;">2x wash in 70% ethanol (each 10 sec)</span>
 
*<span style="font-family:georgia,serif;">90% ethanol</span>
 
*<span style="font-family:georgia,serif;">2x 100% ethanol (very short)</span>
 
*<span style="font-family:georgia,serif;">2x xylene</span>
 
*<span style="font-family:georgia,serif;">Sections are cover slipped with Eukitt</span>
 
 
 
<span style="font-family:georgia,serif;">&nbsp;</span>
 
 
 
=== <span style="font-family:georgia,serif;">Image acquisition</span> ===
 
 
 
<span style="font-family:georgia,serif;">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.</span>
 
 
 
 
 
<div style="clear:none"><span style="font-family:georgia,serif;">[[File:Microscope prp.jpg|frame|left|30px|Olympus Bx52 microscope, equipped with a high-precision motorized MAC5000 LUDL stage and an Optronics MicroFire digital camera.]]</span></div><div style="clear:none"><span style="font-family:georgia,serif;">[[File:Cerebellar-cortex prp.jpg|frame|right|30px|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]]</span></div><div style="clear:none"><span style="font-family:georgia,serif;">[[File:Virtual-slice prp.jpg|frame|center|30px|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]]</span></div>
 
<span style="font-family:georgia,serif;">&nbsp;</span>
 
<div style="clear:both"></div>
 
== <span style="font-family:georgia,serif;">Ontologies and coordinate system</span> ==
 
 
 
<span style="font-family:georgia,serif;">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.</span>
 
 
 
<span style="font-family:georgia,serif;">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.</span>
 
 
 
<span style="font-family:georgia,serif;">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</span>
 
 
 
 
 
 
 
== <span style="font-family:georgia,serif;">Contributing laboratories</span> ==
 
 
 
<span style="font-family:georgia,serif;">Department of Medical Genetics - Jana Boy, Thorsten Schmidt, Ulrike Bichelmeier, Silke Nuber, Olaf Riess<br/>University of Tübingen<br/>Calwerstrasse 7<br/>D-72076 Tübingen<br/>Germany</span>
 
 
 
<span style="font-family:georgia,serif;">Department of Medical Genetics - Carsten Holzmann<br/>University of Rostock<br/>Rembrandtstrasse 16/17<br/>D-18057 Rostock<br/>Germany</span>
 
 
 
<span style="font-family:georgia,serif;">Department of Anatomy - Andreas Wree<br/>University of Rostock<br/>Gertrudenstrasse 9<br/>D - 18057 Rostock<br/>Germany</span>
 
 
 
<span style="font-family:georgia,serif;">Institute for Neurodegenerative Diseases - Stanley Prusiner<br/>University of California<br/>Box 0518<br/>San Francisco<br/>CA 94143-0518<br/>USA</span>
 
 
 
<span style="font-family:georgia,serif;">ZMBH (Zentrum für Molekulare Biologie Heidelberg) – Hermann Bujard<br/>University of Heidelberg<br/>Im Neuenheimer Feld 282<br/>D-69120 Heidelberg<br/>Germany</span>
 
 
 
<span style="font-family:georgia,serif;">Neural Systems and Graphics Computing Laboratory - Trygve B. Leergaard, Francis Odeh, Jan G. Bjaalie<br/>Centre for Molecular Biology and Neuroscience &<br/>Institute of Basic Medical Sciences - Anatomy<br/>University of Oslo<br/>P.O. Box 1105 Blindern<br/>N - 0317 Oslo<br/>Norway<br/>[http://www.nesys.uio.no http://www.nesys.uio.no ]&nbsp;</span>
 
 
 
<span style="font-family:georgia,serif;">[http://www.cmbn.no http://www.cmbn.no]</span>
 
 
 
<span style="font-family:georgia,serif;">&nbsp;</span>
 
 
 
== <span style="font-family:georgia,serif;">Credits</span> ==
 
 
 
<span style="font-family:georgia,serif;">'''PrPpromoter web-application development team:'''<br/>Jan Olav Kjøde<br/>Ivar Andre Moene&nbsp;<br/>Francis Odeh<br/>Trygve B. Leergaard<br/>Jan G. Bjaalie</span>
 
 
 
== <span style="font-family:georgia,serif;">References</span> ==
 
 
 
<span style="font-family:georgia,serif;">'''Boy J, Leergaard TB, Schmidt T, Odeh F, Bichelmeier U, Nuber S, Holzmann C, Wree A, Prusiner SB, Bujard HB, Riess O, Bjaalie JG'''<br/>''Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease-models.&nbsp;''<br/>NeuroImage 2006; 33:449-462</span>
 
 
 
<span style="font-family:georgia,serif;">'''Baron U, Freundlieb S, Gossen M, Bujard H'''<br/>''Co-regulation of two gene activities by tetracycline via a bidirectional promoter.''<br/>Nucleic Acids Res. 1995; 23:3605-3606</span>
 
 
 
<span style="font-family:georgia,serif;">'''Bjaalie JG'''<br/>''Localization in the brain: new solutions emerging.''<br/>Nature Neurosci Reviews 2002; 3:322-325</span>
 
 
 
<span style="font-family:georgia,serif;">'''Bjaalie JG, Leergaard TB'''<br/>''Three-dimensional computerized reconstruction from serial sections: cells populations, regions, and whole brain.''<br/>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)</span>
 
 
 
<span style="font-family:georgia,serif;">'''Cepko C, Ryder E, Fekete DM, Bruhn S'''<br/>''Detection of beta-galactosidase and alkaline phosphatase activities in tissue.''<br/>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</span>
 
 
 
<span style="font-family:georgia,serif;">'''Gossen M, Bujard H'''<br/>''Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.''<br/>Proc Natl Acad Sci USA 1992; 89:5547-5551</span>
 
 
 
<span style="font-family:georgia,serif;">'''Gossen M, et al.'''<br/>''Transcriptional activation by tetracyclines in mammalian cells.''<br/>Science 1995; 268:1766-1769</span>
 
 
 
<span style="font-family:georgia,serif;">'''Holt S, O'Sullivan D'''<br/>''Studies in enzyme cytochemistry I. Principles of cytochemical staining methods.''<br/>Proc R Soc Lond B Biol Sci. 1958; 148:465-480</span>
 
 
 
<span style="font-family:georgia,serif;">'''Paxinos G, Franklin KBJ'''<br/>''The Mouse Brain in Stereotaxic Coordinates''<br/>Academic Press, San Diego 2001</span>
 
  
<span style="font-family:georgia,serif;">'''Schönig K, Bujard H'''<br/>''Generating conditional mouse mutants via tetracycline-controlled gene expression.''<br/>In Transgenic Mouse Methods and Protocols (eds. Hofker,M. & van Deursen,J.) Humana Press, Totowa, NJ. 2003; 69-104</span>
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== Funded by ==
<span style="font-family:georgia,serif;">'''Tremblay P, et al.'''<br/>''Doxycycline control of prion protein transgene expression modulates prion disease in mice.''<br/>Proc Natl Acad Sci USA 1998; 95:12580-12585</span></div><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/>
+
* Fritz Thyssen Stiftung
[[Category:Atlases]]<br/>[[Category:Pages with broken file links|Pages_with_broken_file_links]]
+
* The Deutsche Heredo-Ataxie-Gesellschaft
 +
* The European Union (Marie Curie EST programme, HPMT-CT-2001-00406-03)
 +
* The European Union (QLG3-CT-2001-02256)
 +
* The Research Council of Norway
 +
* The neuroinformatics components of this resource was funded by the Human Brain Project through the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement no. 604102 (HBP)
 +
<span style="font-family:georgia,serif">
 +
== Contact ==
 +
j.g.bjaalie@medisin.uio.no
 +
[[Category:Atlases]]
 +
[[Category:Pages with broken file links|Pages_with_broken_file_links]]

Revision as of 08:14, 14 March 2017

About

The tetracycline-responsive (TetR) gene system is a binary transgenic system in which transgene expression may be activated or silenced by administration of tetracycline, or its derivates (Gossen and Bujard, PNAS 89:5547-5551, 1992; Gossen et al., Science 286:1766-1769, 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. Detailed knowledge about where in the brain gene expression is regulated is essential for the use and interpretation of such models.

The TetOff atlas 1 (PrPpromoter) is an interactive resource providing access to a collection of microscopic brain section from a double transgenic mouse line (Prnp-tTA:βGal), obtained by crossbreeding a tetracycline-responsive prion protein promoter mouse line with a responder mouse line transgenic for a reporter gene construct containing the LacZ reporter gene (encoding the enzyme β-galactosidase).

Access image repository

The virtual microscopy viewer allows interactive zooming and panning. Original images are available for download via separate link. Each section 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 33:449-462, 2006. 

Animal # Genotype Image repository Download original
388.12 prp-lacZ Filmstrip viewer Tiffs

Experimental procedures in brief

Double transgenic mice were produced by crossbreeding a Prnp-tTA mouse line containing the hamster PrP promoter gene (Tremblay et al., PNAS 95:12580-12585,1998) with a responder mouse line transgenic for a bidirectional reporter gene construct containing both the Luciferase and LacZ reporter gene (Baron et al., Nucleic Acids Res. 23:3605-3606, 1995), which encodes the enzyme β-galactosidase. In the resulting double transgenic mice (Prnp-tTA:βGal) 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.

Anaesthetized mice were transcardially perfused with 4% paraformaldehyde. Dissected whole brains were sectioned at 25 µm on a cryostat. Every eighth 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.

High-resolution mosaic section images 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).

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

For further details, see Boy et al. (NeuroImage, 33:449-462, 2006).

Contributing laboratories

  • Department of Medical Genetics, University of Tübingen, Calwerstrasse 7, D-72076 Tübingen, Germany. People: Jana Boy, Thorsten Schmidt, Ulrike Bichelmeier, Silke Nuber, Olaf Riess
  • Department of Medical Genetics, University of Rostock, Rembrandtstrasse 16/17, D-18057 Rostock, Germany. People: Carsten Holzmann
  •  Department of Anatomy, University of Rostock, Gertrudenstrasse 9, D - 18057 Rostock, Germany. People: Andreas Wree
  •  Institute for Neurodegenerative Diseases, University of California, San Franscisco, Box 0518 San Francisco, CA 94143-0518, USA. People: Stanley Prusiner
  •  ZMBH (Zentrum für Molekulare Biologie Heidelberg), University of Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany. People: Hermann Bujard
  •  Neural Systems Laboratory (http://www.nesys.uio.no), Centre for Molecular Biology and Neuroscience & Institute of Basic Medical Sciences, Department of Anatomy, University of Oslo, P.O. Box 1105 Blindern, N - 0317 Oslo, Norway: Histological processing, image acquisition, atlas repository. People: Francis Odeh, Jan O. Kjøde, Ivar A. Moene, Trygve B. Leergaard, Jan G. Bjaalie

Funded by

  • Fritz Thyssen Stiftung
  • The Deutsche Heredo-Ataxie-Gesellschaft
  • The European Union (Marie Curie EST programme, HPMT-CT-2001-00406-03)
  • The European Union (QLG3-CT-2001-02256)
  • The Research Council of Norway
  • The neuroinformatics components of this resource was funded by the Human Brain Project through the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement no. 604102 (HBP)

Contact

j.g.bjaalie@medisin.uio.no