The overall goal of this procedure is to immuno precipitate, cross-linked protein DNA, complexes from dorsal root ganglia tissue following nerve injury. This is accomplished by first inducing sciatic or dorsal column injury. The second step is to cross-link the sample and solubilize the protein DNA complex.
The third step of the procedure is to immuno precipitate a specific protein DNA complex. Finally, the DNA is purified and recovered. Ultimately, results can be obtained that show the enrichment of DNA fragments associated with a protein or DNA histone modification.
This method can help answer key questions in the ronal regeneration field, such as which DNA binding sites, transcription factors and histone modifications are required for the regulation of genes important in promoting axonal regeneration. To begin place an anesthetized animal on the surgical table. Maintain anesthesia throughout surgery through continuous isoflurane oxygen administration via a nose cone before proceeding.
Confirm the correct depth of anesthesia with the toe pinch reflex for sciatic nerve injury. Shave the hind quarter carefully and remove any remaining hair with a depilatory agent. Once the hair is removed, clean the skin with repeated applications of alcohol followed by Betadine.
Make a four millimeter incision through the skin posterior and parallel to the femur. At midt thigh, use fine forceps to spread the muscles apart and expose the white sciatic nerve underneath. Once isolated, the nerve can either be left uncut to serve as a sham surgical control or severed to induce injury.
To finish, pull the skin together and close with two suture clips for dorsal column injury. Remove the hair from the back and clean the incision site as shown previously. Make a 2.5 centimeter incision above the spinal cord from about T seven to T 13.
Use fine forceps to hold the skin while separating the fat. Next, secure the superficial fat with two hooks. Once the fat is pulled back, locate the blood vessel in the space between the sixth and seventh vertebrae to use as a reference point.
Cut the muscle bilaterally over the eighth and 10th vertebrae and insert two hooks to keep it open. Use small scissors to clear the muscles over the laminate at T eight through T 10 holding the spinous process. Perform a laminectomy at T 10 by cutting the connecting bone on both sides.
Carefully lift the top half of the vertebrae away to reveal the spinal cord underneath. Apply a few drops of 2%Xylocaine to anesthetize the spinal cord and then remove the dura mater. Taking care not to touch the spinal cord.
Stopping at this point would provide a sham surgical control to injure the dorsal column. Make a 0.3 to 0.4 millimeter deep cut across the spinal cord. Finally, suture the muscle closed and release the superficial fat.
Pull the skin together and close with suture clips. Place the animal back into the home cage and monitor until fully recovered. 48 hours after injury, euthanize the animal and collect the L four and L five dorsal root ganglia or DRG.
The first step is to expose the ventral spinal column. Next, remove the ventral half of the vertebrae from T 12 to L six. To expose the spinal cord, identify the DRGs located in the space between the vertebrae to the side of the spinal cord.
Use fine forceps to grab the L four and L five DRG lifting slightly. Cut as close as possible to the DRG body. Collect a total of 16 DRGs from four animals and place them into ice cold HBSS.
Briefly centrifuge the DRG add 500 microliters of 1%formaldehyde and incubate the sample for 30 minutes. At 37 degrees Celsius, add 125 millimolar of glycine to stop the fixation and incubate for five minutes at room temperature. After a brief centrifuge aspirate off the buffer and wash the tissue twice with 500 microliters of ice cold PBS plus protease inhibitors cocktail.
Next, aspirate off the PBS and add 400 microliters of SDS lysis, buffer, and disrupt the tissue with approximately 30 strokes of a micro pestle. Then sonicate the chromatin with eight ten second pulses at 70%output. It is suggested that you analyze a sample to confirm DNA fragmentation into lengths of approximately 200 to 1000 base pairs.
Divide the sheared chromatin sample equally into a new tube. For each immunoprecipitation reaction to be performed, bring the volume of each tube up to 500 microliters with chip buffer plus protease inhibitors cocktail. Next, remove five microliters of your diluted sample and transfer into a new tube.
This is your 1%input sample and it will be stored at minus 20 degrees Celsius until needed.Later. Add antibody or normal IgG control to each tube and incubate at four degrees Celsius with rotation overnight. The following day.
Immuno precipitate the complex by adding 30 microliters of protein G magnetic beads and incubating for two hours at four degrees Celsius with rotation. Next place the tube on a magnetic rack to pull down the bound chromatin bead complex. When the solution is clear, carefully remove the supernatant, wash the beads three times with one milliliter of low salt buffer with a three to five minute incubation at four degrees Celsius with rotation for each wash.
Next wash one time with one milliliter of high salt buffer. At this point, retrieve the 1%input sample from minus 20 degrees Celsius and add 150 microliters of chip elution buffer. Set the tube aside at room temperature to be used later.
Returning back to the IP samples, add 150 microliters of one x chip elution buffer to each tube. Incubate the samples at 65 degrees Celsius for 30 minutes with gentle vortexing to elute the chromatin from the beads. Pull down the beads on the magnetic rack and carefully transfer the elute chromatin into new tubes to all tubes including the input samples.
Add 200 millimolar of sodium chloride and 40 micrograms per reaction of protease K and incubate at 65 degrees Celsius for two hours after the incubation, recover the DNA using standard procedures for further analysis here. Semi-quantitative PCR results from DRG tissue following sciatic nerve lesion are shown. Lanes one and two show the PCR signal from the input samples.
The PCR signal in lane four shows that acetylated P 53 binds to the gap 43 proximal promoter region only after injury to the sciatic nerve. No PCR signal is present when the animal receives a sham injury as seen in lane three and when using normal IgG serum shown in lanes five and six. Control PCR from the same DRG tissue shows that acetylated P 53 does not bind to a control region of DNA located in the three-prime untranslated region of the gap 43 gene.
While attempting this procedure, it's important to remember three critical steps for success. Begin with a sufficient amount of tissue, ensure efficient fragmentation and solubilization of the DNA protein complex, and choose a good immunoprecipitation antibody for your protein of interest.
