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DRG injection protocol
Dorsal root ganglia (DRG) are anatomically discrete, neuronal collections located along the vertebral column. Each DRG contains the primary sensory neurons that encode and relay peripheral stimuli to the central nervous system (CNS) from specific body regions.We present here a protocol that uses a DRG injection with a viral vector in the mouse. The experimental process of stereotactic injection into dorsal root ganglion DRG (Take the 5th dorsal root ganglion of the left lumbar vertebra as an example) of the mouse is as follows.
Prepping mice for surgery
● Anesthetize the mouse with intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg). Wait until the mouse becomes motionless (~5 min).
● Remove the fur around the mouse’s dorsal spine using electric shaver for small animals, then sterilize the skin surface on the back with iodophor and 75 % ethanol in turn.
● Make a 1.5-cm-long skin incision along 1 cm proximal to superior location of median iliac spine on the back of the mouse, a similar-sized incision was made on the fascia and muscles to the left of the spine starting from the erector spinae.
● The muscles and spine were separated by blunt dissection using tip-curved forceps, scrape off the muscle and fascia from the spinal vertebral laminae using a scalpel.
● Then stop the bleeding and remove the blood by using sterilized cotton buds or surgical absorbable sponges to allow clear visualization, exposing the vertebral lamina, articular process and transverse process, to the corresponding region of DRG of the L4- L5 lumbar vertebrae in the superior location of the iliac spine.
Target coordinates
Mounting the spine of the mouse to the stereotaxic apparatus, use a skull drill to lightly drill the edge of the articular process and the vertebral lamina and gently lift the drilled vertebral lamina and articular process with pointed forceps under the microscope, then the L4 and L5 spinal ganglia adjacent to the spinal cord can be visible.
Equipment setup
● Once enough of the DRG has been exposed for injection, install the micro-injection pump and glass electrode to keep them be at a 45° angle as close to tangential with the injection surface as possible, which would facilitate a large spread of the drug or virus.
● Two injection points can be selected on the DRG and entry distance of the glass electrode tip is 0.2-0.3 mm.
● Inject the virus into each DRG at a rate of 30 nl/min to a total volume of 200 nl–500 nl using a syringe pump and stop to retain the needle for 5 minutes.
Post-injection care
● After the injection is finished, use 4-0 sutures to suture muscles and skin on the back layer by layer, and use iodophor to sterilize the incision.
● Keep the mouse in a warm environment until it wakes up and place it back in its cage for normal breeding.
● At least 4 weeks after successful delivery, DRG was taken and used for immunohistochemistry to detect virus infection and knockout effect.
NOTE: The methods of virus injection in various site are similar, just alter the injection dosage according to the characteristics and titer of different viruses. It is recommended to conduct pre-experiment before using each virus.

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