AAV-Mediated Gene Delivery for Visualization of Compromised Retinal Membrane Integrity

Begin with the eye of a transgenic mouse previously injected with an adeno-associated virus (AAV) carrying the gene of interest that encodes a fluorescent protein.

The mouse lacks a gene essential for maintaining the integrity of the inner limiting membrane (ILM), a barrier that separates the retina from the vitreous cavity.

Loss of this gene mimics retinal degeneration, allowing the virus to penetrate deeper into the retinal cell layers.

Infected retinal cells express the gene of interest, producing fluorescent proteins, allowing them to fluoresce.

Now, dissect the eye and remove the cornea and lens.

Carefully detach the retina from the retinal pigment epithelium (RPE) and the sclera, the eye’s outer layer.

Immerse the tissue in fixative.

Wash with phosphate buffer to remove residual fixative.

When observed under a confocal microscope, fluorescence in the retinal cells indicates successful viral penetration through the compromised inner limiting membrane.

For retinal flat mounts facilitate the dissection by fixing the enucleated eyes in 4% paraformaldehyde for 15 to 30 minutes. This fixation and the fixation following the dissection must be performed properly as this is critical to preserve the GFP fluorescence. Within 15 to 30 minutes of the fixation period. Dissect the eyes, remove the cornea and lens.

Separate the retina from the retinal pigment epithelium and from the sclera by cutting around the ora serrata and the optic nerve. After the dissection, immerse the retinas in 4% paraformaldehyde for another 30 minutes to fix the tissue and the fluorescent protein in transduced retinal cells. After the fixation, wash the specimen for five minutes in sterile PBS at pH 7.4.