Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The overall goal of this procedure is to deliver a target gene to the retinal pigment epithelium in a living mouse. This is accomplished by first opening the eyelid and protruding the eyeball to expose the equator. The next steps are to make a small hole posterior to the limbus.

Inject the viral vectors into the subretinal space from a blunted needle and observe the formation of a subretinal bleb. Ultimately, immunofluorescence microscopy is used to show the expression of fluorescence markers delivered by the injected virus in the retinal pigment epithelium. This is an EEG and convenient technique for subretinal injection of viral vector to retinal pigment.

Epithelio Visual demonstration of this method is critical as the subretinal injection test are difficult to lung. For the beginners. First, prepare a microliter syringe with a 33 gauge blunt needle sterilize at 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 two microliters ready to inject with the viral vector injection.

Prepared anesthetize adult mouse with an intraperitoneal injection of lenta diazepam and slaine confirmed 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%ide 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 temporally placed or nasally placed. Holes are not good choices as they're hard to cover. While puncturing, do not hit the limbal vessel or the lens and do not insert the entire battle 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 To not penetrate the scleral tissue with excessive pressure because the needle should be placed in the potential subretinal space. If the needle punctures the cleal tissue with the 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 widely important. Then withdraw the needle just as gently. Now the eyeball can be released.

Subretinal blabbing indicative of vector accumulation should be visible. Finally, gently close the eyelid to cover the injection site. Self-sealing of the hole will occur.

To evaluate the gene delivery, sacrifice the mice using carbon dioxide asphyxiation, and after euthanasia has been confirmed. Nucleate the eyes using scissors. Fix the eyes in 4%PFA at four degree Celsius for at least an hour, but preferably a few days later.

Grip and puncture the cornea using forceps and micro scissors. Then cu along the limbus to remove the cornea following removal of the cornea. Use forceps to remove the lens.

Next, trim the ciliary body. Then pull the whole retina from the RPE Choroid Sclera complex following removal of the retina. Cut the complex radially from the periphery to the center near the optic nerve to make four to eight pedal like shapes and make the structure flat for mounting.

Now, flip the complex over RPE side down and trim off any remaining tissues on the scleral side, including muscles, conjunctiva and optic nerve. This makes a flatter RPE Cort scleral complex flat mount on a glass slide. Absorb the excess PBS with an absorbent sponge.

Apply about 30 microliters of mounting media and cover slip the complex. Store the slide at four degrees Celsius when not being viewed. Using the described protocol commercially available LV vector with CMV promoters expressing both GFP and RFP were injected.

The eyes were enucleated after 10 weeks and RPE flat mount slides were prepared. Signal intensities of GFP and RFP varied between the RPE cells at the injection site, and transduction was limited to the area where the injection blood formed. Increasing the magnification from four DX to 400 x made it possible to evaluate the pathology of the I with even more resolution.

Although not shown here, the transduced cells expressed vector even 36 weeks after the injection. One mastered. This technique can be done in five minutes if it is performed properly.

While attempting this procedure, it is important to remember to place the needle or creatively and ly into the subretinal space After its development. This technique paved the way for the researchers in the field of ophthalmology to explore gene therapy for treatment in birth, monogenic inherited pathy and age related macular degeneration.