This article describes a protocol for intrathecal delivery of adeno-associated viruses (AAV) in mice, aimed at enabling targeted gene expression. The method involves precise anatomical localization and injection techniques to ensure effective delivery of the viral vector into the cerebrospinal fluid.
Fill a syringe with a solution containing adeno-associated viruses, or AAV, and lidocaine, a local anesthetic.
Place an awake mouse in a prone position and cover its upper body.
Hold the mouse's pelvic girdle and shave its lower back to expose the skin. Sterilize it with iodine and ethanol.
Locate the gap in the lower spine and gently press to mark it.
Rotate the tail to align the spine and insert the syringe vertically.
Adjust the angle until a tail flick occurs, confirming needle entry into the intrathecal space where the cerebrospinal fluid or CSF flows.
Slowly inject the solution for even distribution.
The viral particles bind to neurons, enter inside, and release their genetic material into the nucleus, enabling targeted gene expression.
Lidocaine temporarily blocks nerve signals, causing short-term leg paralysis, confirming successful delivery in the CSF.
This protocol establishes intrathecal delivery as an effective method for treating CNS diseases.
Before beginning the procedure, attach a 27-gauge needle to a 25-microliter Hamilton syringe and align the beveled tip of the needle with the volumetric scale on the syringe, then carefully load 8 microliters of 4 times 10 to the 10th genome copies of the virus solution into the syringe, taking care to avoid bubbles.
Next, place an awake 30 to 70 day old, 12 to 30 gram mouse on a bed piece in the prone position in a biosafety hood and cover the upper body with sterile gauze to calm the mouse and to avoid being bitten. Firmly grip the mouse on its pelvic girdle, with a thumb on one side and the forefinger and middle finger on the other side, keeping the skin between the bilateral pelvic girdles taut with the thumb and forefinger, then shave the fur between the bilateral pelvic girdles and sterilize the exposed skin with an iodide-based scrub and 70% ethanol.
For direct intrathecal delivery of the adeno-associated virus solution, palpate the intervertebral space along the midline between the bilateral pelvic girdles and use a fingernail to depress an indentation into the skin to indicate the L5 to L6 intervertebral space. Rotate the base of the tail slightly and gently to reveal the midline of the spine and adjust the bevel of the needle toward the head of the animal. With the mouse fixed firmly into position, align the needle along the midline of the spine and insert the needle gently and vertically into the center of the indentation, keeping the syringe in a central sagittal plane.
When the needle comes into contact with the bone, slowly decrease the angle to approximately 30 degrees and slip the needle into the intervertebral space. A sudden tail flick is a sign of a successful entry into the intradural space. When the needle enters the intervertebral space, the tip will feel firmly clamped. Inject the vector solution and maintain the needle within the intradural space for one minute, then slowly withdraw the needle with rotation to minimize leakage.
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