Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

JoVE, 2011 · DOI: 10.3791/2803 · Published: July 18, 2011

Simple Explanation

The study focuses on the dorsal root ganglia (DRG) as an injury model system because it innervates both the central and peripheral nervous systems. Injury to nerve fibers in the PNS reveals transcriptional programs distinct from those in the CNS. The described ChIP protocol uses fixed mouse DRG tissue following axonal injury to characterize the pro-regeneration chromatin environment. The protocol involves sciatic and dorsal column nerve injury, cross-linking, nuclei preparation and chromatin shearing, immunoprecipitation, washing, elution, and DNA recovery.

Study Duration

Not specified

Participants

4 mice (2 injured and 2 sham DRG from each animal)

Evidence Level

Not specified

Key Findings

1
The protocol demonstrates fragmented DNA to a length of approximately 200-1000 bp following sonication.
2
Acetylated p53 binds to the GAP-43 proximal promoter region 48 hours after injury to the sciatic nerve.
3
No PCR signal is present when the animal receives a sham injury only, and when using normal IgG serum for the IP.

Research Summary

This protocol combines the DRG injury model with chromatin immunoprecipitation to investigate the transcriptional and epigenetic environment after injury to either the PNS or CNS. The ChIP protocol is useful for characterizing putative binding sites for transcription factors and determining occupancy in response to injury. The protocol outlines critical steps such as efficient fragmentation and solubilization of the DNA-protein complex and the availability of a good immunoprecipitation antibody.

Practical Implications

Study of Transcription Factor Binding

The method can be used to characterize putative binding sites for transcription factors.

Analysis of Epigenetic Modifications

Epigenetic modifications of histones and DNA at transcription factor binding sites can be monitored.

High-Throughput Analysis

The protocol can be adapted for ChIP-on-chip assays to increase the number of identified transcription sites.

Study Limitations

1
Low level of starting material due to small amount of DRG tissue.
2
Heterogeneous IP signals due to the mixed population of neurons and glial cells in DRG.
3
Validation needed for ChIP in DRGs if using fluorescent activated cell sorting or immuno-isolation of neurons.

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