Gene Transfer via Posterior Semicircular Canal Injection

To perform gene transfer experiments in vivo, P1 pups were anesthetized and injected via the posterior semicircular canal (PSCC) technique as previously described.⁶² (link) Briefly, injection was performed using beveled glass microinjection pipettes, which were pulled from capillary glass on a P-2000 pipette puller (Sutter Instruments). Pups were anesthetized by rapid induction of hypothermia for 3–4 min on ice until loss of consciousness, and this state was maintained on a cooling platform for 10–15 min during the surgery. The surgical site was disinfected by scrubbing with Betadine and wiping with 70% ethanol. A postauricular incision was made to expose the PSCC and penetrate the tip of the micropipette. A total volume of 1 μL of either virus was unilaterally introduced at a rate of 300 nL/min into the left ear. The skin incision was closed using superglue. Body temperature was maintained on a 37°C warming pad for 30 min after surgery and before reintroduction into the parental cage.