This article describes a method for reprogramming glial cells into neurons using adeno-associated viruses in a transgenic mouse model. The procedure involves precise stereotaxic injections to deliver viral vectors that activate neuronal reprogramming genes.
Secure an anesthetized transgenic mouse in a stereotaxic frame with its skull exposed.
This mouse expresses Cre recombinase in glial cells.
Position the stereotaxic arm holding the capillary tube over the skull and align it above the bregma.
Adjust the stereotactic axes for precise targeting.
Drill a hole at the target site, ensuring the dura mater remains intact. Remove bone fragments.
Flush the capillary tube with buffer. Draw up an air bubble, then load the adeno-associated virus carrying inactive neuronal reprogramming and fluorescent protein genes.
Lower the capillary tube to the target depth and inject the viral solution into the glial cell-rich region.
Carefully retract the tube to prevent backflow.
Close the incision and allow recovery.
The internalized viruses deliver loxP-flanked inactivated genes to the glial nucleus. Cre recombinase flips them, activating neuronal reprogramming genes and driving neuronal conversion. Reprogrammed interneurons express the fluorescent protein under the neuron-specific promoter.
To inject reprogramming factors, place an anesthetized mouse in the stereotaxic frame, administer appropriate analgesia at the beginning of the surgery, then bring the tip of the glass capillary of the injection needle just above bregma. Make sure the capillary tip is perfectly straight in both A-P and M-L planes. Reset both the M-L and A-P values to 0.0 on the digital coordinate counter.
To make sure the head of the animal is in a perfectly flat position, use the digital coordinate counter to measure the D-V coordinate value when the A-P arm is at plus 2.0 and minus 2.0, as well as when the M-L arm is at plus 2.0 and minus 2.0. Adjust the height of the tooth bar and ear bars accordingly. Afterward, raise the syringe slightly and drill a hole using a dental drill at the injection coordinates.
Start to drill at the site, working in a circular and gentle manner. Then, place a piece of cotton gauze over the open incision and flush the syringe with saline solution. After flushing, draw up an air bubble and then 1 microliter of solution containing the viral vector. Make sure that the viral solution can be easily visualized below the air bubble.
Next, lower the syringe, progressing slowly to the desired depth and be sure that the trajectory is clear of bone fragments. Subsequently, inject 1 microliter of the viral solution at a rate of 0.4 microliters per minute. When the injection is done, allow two minutes for diffusion before syringe withdrawal.
After diffusion, slowly retract the syringe until the tip of the capillary is completely withdrawn from the brain. Then, carefully suture the incision and remove the animal from the stereotaxic frame. Monitor the animal in a postoperative station until consciousness is regained.
Animals are kept for up to 12 weeks after virus injection to allow resident glia to reprogram into mature neurons.
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