This article outlines a method for genetically modifying neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ) of the mouse brain using viral vectors. The procedure involves precise stereotaxic coordinates to ensure accurate targeting and minimal damage to surrounding tissues.
Begin with an anesthetized mouse with an exposed skull in a stereotaxic frame.
Lower the syringe holder until the needle tip aligns with the bregma, a skull reference point.
Using the pre-determined stereotaxic coordinates, locate the ventricular-subventricular zone or
V-SVZ, a neurogenic niche primarily containing neural stem cells or NSCs, and mark this region.
Remove the syringe and carefully drill a small hole, avoiding brain damage.
Fill a sterile syringe with viruses carrying the transgene and position the needle over the brain surface.
Reposition the syringe and lower it until the tip touches the innermost brain surface layer.
Penetrate the brain to reach the V-SVZ, slowly inject the virus, hold briefly, and then withdraw the syringe.
In the V-SVZ, the virus enters NSCs via membrane fusion. Its RNA reverse transcribes into DNA.
This DNA then integrates into the NSC genome, enabling stable genetic modification of NSCs.
After anesthetizing, a six to eight week old mouse shave the area between its ears. Next, disinfect the skin using an iodophor such as iodine povidone or 70% ethanol. Then, place the animal in a prone position on a stereotaxic frame.
Carefully fix its head using the ear bars and the palate support of the apparatus. Keep the mouse on a heating pad set at 37 degrees Celsius and apply ophthalmic lubricant to its eyes.
Next, make a 1-centimeter long incision on the scalp and gently retract the skin to expose the skull. Carefully, clean the bone surface with a sterile cotton tipped applicator and remove any remaining tissue. After that, mount the sterilized syringe on the stereotactic device using the syringe holder. Position the needle on the bregma and ensure that the 0 position of the dorsal ventral axis is at the skull surface at the bregma.
Now, move the syringe to the x and y designated coordinates, then annotate the x, y, and z destination coordinates in the Vernier scale as the injection site, and mark the bone using a surgical marker pen.
The Vernier scale is an accurate measuring tool, allowing us to determine the stereotactic coordinates from bregma with high precision. The 0 on the main scale marks the units, and the number aligned with the main scale determines tenths of a millimeter.
Afterward, move the syringe away from the working area. Carefully drill a hole on the skull without damaging the brain.
The drill must be used horizontally to the skull surface, and continuous monitoring of the advances must be made in order to stop drilling once the pial surface is exposed. Do not damage the underlying brain.
Then, load the syringe with a 33-gauge sharp beveled needle with 1 microliter of the viral solution. Position the syringe needle at a 90-degree angle to the brain surface. Move the syringe back to the injection site and lower it until the tip touches the pial surface and penetrates the brain.
To minimize damage to the brain tissue due to excessive fluid pressure, slowly release the viral suspension. After the injection, wait for 5 to 10 minutes to minimize the backflow of viral suspension before slowly retracting the syringe.
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