Name: chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury
Uploaded: 2011-07-20T12:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury at JoVE.com

We would like to thank Andrea Tedeschi for help in setting the initial ChIP experiments in the laboratory and Ricco Lindner
 for his contribution to fine tune the conditions for ChIP. This work was supported by the Hertie Foundation; the Fortune Grant, University of 
 Tuebingen, and the DFG DI 1497/1-1 grants (all granted to Simone Di Giovanni).

1. Sciatic &amp; dorsal column nerve injury
<ol>
 <li>The animal is placed on a surgical towel and underneath it a thermopad is present throughout the procedure keeping the body temperature of the mouse at 37&deg;C. All animals are anesthetized for surgery with a continuous isoflurane/O2 administration. Surgical instruments are autoclaved before the procedure.</li>
 <li>For sciatic injury, both hindquarters are carefully shaved, and depilation is completed with generic hair remover prior to cleansing skin wi...

<ol>
	<li>Beirowski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+analysis+of+Wallerian+degeneration+using+restricted+axonal+labelling+in+YFP-H+mice.">Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice.</a> Journal of Neuroscience Methods. 134, 23-35 (2004).</li><li>Carey, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatin immunoprecipitation (ChIP). , (2009).</li><li>Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+micro+chromatin+immunoprecipitation+assay+(&amp;mu;ChIP.">A rapid micro chromatin immunoprecipitation assay (&amp;mu;ChIP.</a> Nat. Protoc. 3, 1032-1045 (2008).</li><li>Haring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.+Plant+Methods.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods.</a> 11, 3-11 (2007).</li><li>Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nat Neurosci. 8, 723-729 (2005).</li><li>Makwana, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+in+successful+peripheral+regeneration.">Molecular mechanisms in successful peripheral regeneration.</a> Febs J. 272, 2628-2638 (2005).</li><li>Tedeschi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+p53-CBP/p300+transcription+module+is+required+for+GAP-43+expression,+axon+outgrowth,+and+regeneration.">A p53-CBP/p300 transcription module is required for GAP-43 expression, axon outgrowth, and regeneration.</a> Cell Death and Differentiation. 16, 543-554 (2009).</li><li>Teng, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+regeneration+in+adult+CNS+neurons--signaling+molecules+and+pathways.">Axonal regeneration in adult CNS neurons--signaling molecules and pathways.</a> J Neurochem. 96, 1501-1508 (2006).</li></ol>

This protocol provides a method to directly ask about the chromatin environment during axonal regeneration in the adult 
nervous system following axonal injury. It incorporates the DRG injury model with chromatin immunoprecipitation to probe the transcriptional and 
epigenetic environment subsequent to injury to either the PNS or CNS. It is particularly useful for investigators who would like to characterize 
putative binding sites for their favorite transcription factor, and to determine whether the occupancy of these s...

<table border="1">
	<tbody>
		<tr>
			<th>Reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>10x ChIP Buffer</td>
			<td>Cell Signaling</td>
			<td>7008</td>
		</tr>
		<tr>
			<td>2x ChIP Elution Buffer</td>
			<td>Cell Signaling</td>
			<td>7009</td>
		</tr>
		<tr>
			<td>ChIP Grade Protein G Magnetic Beads</td>
			<td>Cell Signaling</td>
			<td>9006</td>
		</tr>
		<tr>
			<td>Magna Grip Rack (8 well)</td>
			<td>Millipore</td>
			<td>20-400</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>MERCK</td>
			<td>UN 1888</td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>ROTH</td>
			<td>CP10.1</td>
		</tr>
		<tr>
			<td>10x Glycine Solution</td>
			<td>Cell Signaling</td>
			<td>7005</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Sigma</td>
			<td>G1767</td>
		</tr>
		<tr>
			<td>10x HBSS</td>
			<td>Gibco</td>
			<td>14185</td>
		</tr>
		<tr>
			<td>Histone H3 antibody (rabbit)</td>
			<td>Cell Signaling</td>
			<td>2650</td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG</td>
			<td>Cell Signaling</td>
			<td>2729</td>
		</tr>
		<tr>
			<td>Phenol/Chloroform/Isoamyl Alcohol</td>
			<td>ROTH</td>
			<td>A156.1</td>
		</tr>
		<tr>
			<td>Protease Inhibitors Cocktail Tablets</td>
			<td>Roche</td>
			<td>04 693 116 001</td>
		</tr>
		<tr>
			<td>Proteinase K (20 mg/ml)</td>
			<td>Cell Signaling</td>
			<td>10012</td>
		</tr>
		<tr>
			<td>SDS Lysis Buffer</td>
			<td>Upstate</td>
			<td>20-163</td>
		</tr>
	</tbody>
</table>
<table border="1">
	<tbody>
		<tr>
			<th>Equipment needed</th>
		</tr>
		<tr>
			<td>Sonicator</td>
		</tr>
		<tr>
			<td>Micropestle</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury at JoVE.com

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Research

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