Subretinal Injection of Viral Vectors into the Retinal Pigment Epithelium in a Mouse Model

Begin with an anesthetized mouse and apply a pupil-dilating agent.

Under a microscope, open the eyelid and slightly protrude the eye to expose the injection site.

Apply a drop of ophthalmic viscoelastic solution, then place a coverslip to visualize the retina.

Using a fine needle, create a hole behind the cornea-sclera junction, avoiding damage to the blood vessels and the lens.

Through this pre-punctured hole, insert a blunt syringe tangentially into the subretinal space near the retinal pigment epithelium or RPE, which supports retinal neurons.

Inject the lentiviral vector carrying target genes into the subretinal space.

Observe the formation of a subretinal bleb due to fluid accumulation, confirming successful vector delivery.

Gently close the eyelid to cover the injection site and allow the mouse to recover.

The lentivirus enters the RPE via endocytosis, releases its genetic material, and converts it into the corresponding DNA, which integrates with the host genome, enabling gene expression.

First, prepare a microliter syringe with a 33-gauge blunt needle. Sterilize it using ethylene oxide gas. Next, prepare a dilution of the viral vectors in PBS in a micro fuge tube at about 1 million titer units per microliter. Now, when loading the syringe, flush it several times with the solution to remove any dead space in the syringe. Get 1.5 to 2 microliters ready to inject.

With the viral vector injection prepared, anesthetize an adult mouse with an intraperitoneal injection of tiletamine, zolazepam, and xylazine. Confirm that the animal has been anesthetized by the absence of reflexes. Once anesthesia has been confirmed, dilate its pupils with a drop of 0.5% phenylephrine and 0.5% tropicamide. Now, under an operating microscope, open the eyelid and protrude the eye to expose the equator. Hold the eye in this position by placing your fingers outside the orbital rim.

Next, apply ophthalmic viscoelastic solution to the corneal surface and place a small round cover slide over the cornea. Thus, the retina can be viewed. Now, slightly posterior to the limbus, and inferiorly in the right eye, or superiorly in the left eye, use a sterile 30-gauge half needle to make a small hole. Temporarily placed or nasally placed holes are not good choices as they are hard to cover.

While puncturing, do not hit the limbal vessel or the lens, and do not insert the entire bevel of the needle.

For the subretinal injections, insert the vector injection needle through the hole at a 45-degree angle to the iris plane. Push the needle posteriorly toward the peripapillary area, following the demarked path to cross the vitreous cavity. Approach the needle into the subretinal space until mild resistance is felt, which indicates that the RPE layer has been reached.

Do not penetrate the scleral tissue with excessive pressure because the needle should be placed in the potential subretinal space. If the needle puncture the skull tissue with excessive power, it enters into the extraocular orbital space without resistance.

Now, gently inject the viral vector into the subretinal space. Keep the needle very still to prevent tissue damage, which is vitally important. Then, withdraw the needle just as gently. Now, the eyeball can be released. Subretinal blebbing indicative of vector accumulation should be visible. Finally, gently close the eyelid to cover the injection site. Self-sealing of the hole will occur.