Spatially-Restricted Gene Transduction in Mice

Bilateral injection of rAAVs into the spinal parenchyma of adult mice allows for spatially- and temporally-restricted, stable gene transduction without causing persistent inflammation, tissue injury, or glial scar formation^{6 (link),10 (link),87 (link)}. Mice were anesthetized with a mixture of fentanyl (50 µg/kg), midazolam (5 mg/kg), and medetomidine (0.5 mg/kg), and laminectomy was performed. Using a microprocessor-controlled minipump and a 35 gauge bevelled “NanoFil” needle (World Precision Instruments), 500 nl of a 2:1 mixture of rAAV stock with 20% mannitol were injected into the parenchyma of the spinal cord dorsal horn of the L3-L5 segments on each side (total of two injections per mouse) at a flow rate of 100 nl/min^{6 (link),10 (link),87 (link)}. Anesthesia was reversed using a mixture of atipamezole (0.75 mg/kg), flumazenil (0.5 mg/kg), and naloxon (1.2 mg/kg), and the animals placed overnight on a heating plate set to 39 °C. Mice were allowed to recover for a minimum of 3 weeks after surgery before further analysis to allow for transgene expression. After experiments, mice were sacrificed, and correct delivery and expression or functionality of the injected rAAVs was assessed.