The overall goal of this procedure is to establish an in vitro model for studying mammalian axon regeneration using adult dorsal root ganglion neurons or DRG neurons. This is accomplished by first dissecting out the DRGs from adult mice, which are then dissociated into single cells. Then the dissociated neurons are transfected by electroporation and cultured for a few days to genetically manipulate their gene expression.
The final step is to resuspend and reflate the neurons for additional overnight culture. Ultimately, microscopy based axon length analysis is used to study axon regrowth from genetically manipulated neurons. The main advantage of cell resus suspension and repeating step used in this technique is that it allows analysis of axon growth from neurons that have already been genetically manipulated.
This technique is particularly useful for loss of function Studies in which RNI approach is used to downregulate gene of interest demonstrating the procedure will be Dr.Sfu, a postdoc fellow from my lab. To prepare the neuronal culture cover slips first, bathe them in 10%hydrochloric acid overnight. The next day sonic heat them in distilled water for an hour.
In three 20 minute segments, store the cleaned cover slips in 70%ethanol and allow them to air dry before use. Next to coat the dried cover slips. Set up a multi-well plate with one slip and 100 microliters of coating solution per well.
Incubate the plates without any agitation for one to two hours at 37 degrees Celsius. After the incubation, remove the coating solution and wash the cover Slips three times with sterile one XPBS. After sacrificing a fully anesthetized six to 10 week old adult mouse, unresponsive to painful sensations.
First, remove its dorsal skin. Second, cut free all the vertebrae from the neck to the tail, including the attached tissues. Finally, remove any internal organs attached to the inside of the tissue.
Rinse the removed spinal column with one XPBS two to three times, then pin it to a dissecting plate ventral side up. Carefully remove the muscles to expose the sensory nerves. The nerves connected to DRGs at the lumbar level are the thickest and are easily located.
Therefore, for most experiments, only the lumbar DRGs are harvested. Cut through each vertebrae along the center line with small scissors and carefully remove the intervertebral discs to isolate the vertebrae. Beginning at L one, use forceps to split each vertebrae and dissect out the lumbar DRGs.
Locate the DRGs with forceps by tracing each lumbar nerve towards the spinal cord. Repeat this process for L two to L six. Next, use spring scissors to trim the peripheral nerve dorsal and ventral roots off the isolated DRGs.
Store the DRGs in a chilled 1.5 milliliter fuge tube filled with em EM medium if necessary. The same procedure can be used to harvest DRGs from other spinal levels. After collecting all DRGs, replace the MEM medium with one milliliter, collagenase a solution and incubate the DRGs at 37 degrees Celsius for 90 minutes.
Next, replace the collagenase a solution with 500 microliters of one x triple E express digestion solution and return the tubes to the incubator for another 15 to 20 minutes after the second digestion. Wash the DRGs by replacing the solution with one milliliter of prepared medium containing 5%FBS, swirl the DRGs and allow them to settle. Repeat this wash.
Step twice before proceeding. After removing the last wash, add 600 microliters of culture medium and gently pipette up and down to tri the tissues. Do this 20 to 30 times using a 1000 microliter pipette tip after tri allowed the non dissociated tissues to settle down to the bottom of the micro fuge tube.
Transfer only the cell suspension to a 10 milliliter sterile tube and add another 600 microliters of culture medium. Repeat the tri step until most of the tissues are dissociated. The suspension contains both neurons and non neuronal cells.
Typically, about 50, 000 cells are collected from six dgs enough for one electroporation reaction, centrifuge the dissociated cells of six dgs and discard as much supernatant as possible. Then resuspend the cells in 100 microliters of transfection solution containing DNA plasmid or SI RNA with three to four gentle strokes through a 200 microliter pipette tip. Transfer the suspension to an electroporation, vete, and electroporated the cells after the electroporation.
Immediately add 500 microliters of warm culture medium containing FBS to the vete. For most experiments requiring Resus suspension and replating culture, the neurons on a plastic culture dish at 10 to 20, 000 cells per well. However, to examine axon growth directly after electroporation, the neurons can be plated directly onto coated glass Cover slips at three to 5, 000 cells per well.
After four hours of cell culture, the neurons will have attached to the substrate. Gently replace the medium tainted with transfection solution with 500 microliters of fresh warmed medium and return the plates to the incubator. Analysis of gene expression from DNA plasmids is feasible just a few hours after electroporation.
However, to analyze downregulation of gene expression in new axonal growth, longer culturing time is required. After three days of culture on the high density plates, replace the medium with one milliliter of prewarm fresh medium and flush the neurons into solution with six to 10 gentle strokes of the pipette. Then non neuronal cells will stay attached to the plate, transfer the resuspended neurons to a micro fuge tube and gently T them 10 to 15 times to dissociate the cell clumps.
To ensure easier use suspension of attached neuron after three days in culture, the coating of the culture dish before the initial culture is limited to just 1, 2, 3 hours. During the S suspension steps, we usually check the cells constantly under an inverted microscopes to observe if the cells are detached from the cultural dish Plate. The neurons onto newly prepared cover slips at a low density the next day, analyze the new axonal growths.
In the absence of any added extracellular growth factors, the adult DRG neurons usually start to grow axons. 48 hours after first plating the axons often show branched morphologies. In contrast, the replated neurons start to extend axons only a few hours after plating and the axons elongate with reduced branching.
These results suggest that replated neurons share similar properties to those of regenerating neurons in vivo. By using this approach, a loss of function study was used to examine the role of axon regeneration associated transcription factors see JU in axon growth from adult DRG neurons. In vitro.
The results showed that electroporation of a group of four different irna targeting different regions of cgen markedly reduce the protein levels of cgen in adult DRG neurons. Three days after transfection, when the neurons were replated and cultured overnight axon growth from cgen knockdown neurons were significantly reduced. These results indicate that cultured adult DRG neurons provide a useful model system to study axon growth from adult neurons.
After watching this video, you should have a good understanding of how to dissect transfect and culture. Adult must also root ganglion neurons for studying membrane accident regeneration in vitro.
