Name: subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye
Uploaded: 2012-11-25T14:00:00.000+00:00
Duration: 5 min 9 s
Description: Watch this Scientific Journal Video about Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye at JoVE.com

Research to Prevent Blindness; Experimental assistance from Takayuki Nagasaki; This research complies with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. KJW is supported by NIH grants 5T32EY013933 and 5T32DK007647-20. VBM is supported by NIH grant K08EY020530.

1. Assemble Devices for the Subretinal Injection
<ol>
 <li>Purchase or make a 100 &mu;m diameter needle from a glass capillary tube. This can be done manually by using a Sutter P-97 pipet puller or other similar equipment. The end of the capillary tube will be heated and pulled until it reaches the desired diameter (100 &mu;m). A smaller diameter needle can be used for gene therapy vectors; however, this is the recommended diameter for cell injection without damage to the cells or the eye. Compared to steel needles, the glass capillary needle has a very fine, but blunt tip, to allow for visualization of the injection fluid and access to the subretinal space without causing retinal breakage. Clean these pulled needles if needed using 70% ethanol, and store in a sterile container. A 15 cm sterile tissue culture dish with tape to hold the needles in place is one way in which to keep the injection needles covered and in a sterile environment. At this point, complete the set-up within the surgical room where the procedure will take place. It is easiest to keep all equipment in the surgical room at all times in order for it to remain sterile, especially if this procedure is done within a barrier facility.</li>
 <li>Aspirate silicone into the injection needle and eject the silicone to leave a coating along the inner walls of the needle. The silicone coating maximizes the passage of cells and viruses through the needle bore, which would otherwise stick to the inner glass capillary walls.</li>
 <li>Obtain an approximately 8 cm length of sterile plastic tubing by cutting a 25 &frac34;-gauge blood collection set with luer-lock at the point directly after the needle (removing the needle from the tubing).</li>
 <li>Fill a 1 ml Sub-Q 26 5/8-gauge slip-tip syringe with sterile saline and remove the needle from the syringe. </li>
 <li>Attach the injection needle to the cut end of the plastic tubing and attach the syringe to the luer-lock at the other end. </li>
 <li>Aspirate sterile saline to fill the dead space in the tubing and capillary needle, ejecting some saline through the needle to ensure that it is correctly attached without any leakage. Next, aspirate a small, approximately 1 &mu;l amount of air, into the tip of the injection needle. This will separate the saline from the injection fluid and provide a visual cue indicating the end of the injection fluid during the surgical procedure. The injection apparatus should be kept on a clean surface wiped down with 70% ethanol during the surgical procedure. Additionally, all equipment used during the surgical procedure should be cleaned both before and after with 70% ethanol, for the exception of the injection needles which should already be kept sterile and ready for use. These needles should be disposed of after surgery, and not re-used.</li>
 <li>Finally, aspirate injection solution containing the desired packaged gene therapy viral vector or stem cells. This can be done by taking up the injection solution into a sterile pipet tip, then pipetting the solution onto the surface of a sterile cell culture dish. The solution can now be easily aspirated into the injection needle using the syringe. The amount needed will vary depending on the age of the mouse. For our purposes we use post-natal day 5 mice, which can be injected with 0.5-0.8 &mu;l of injection fluid. Smaller amounts, 0.5 &mu;l or less, can be used for younger mice and larger amounts, such as 1 &mu;l, can be used for adult mice. The viral titer or number of cells needs to be determined based on the virus/cells being injected and their desired target tissue, as too many viral particles or cells in a small volume can lead to toxicity for the retinal cells. In the video, for visualization purposes, we have used 1.5 &mu;l, a greater amount of dye than necessary to inject into the eye. </li>
</ol>
2. Accessing the Subretinal Space
<ol>
 <li>Within the sterile surgical room, stabilize or anesthetize the mouse according to the locally approved animal handling protocol, which will vary by age of the mouse. Common anesthesia for adult mice includes the use of isoflurane gas or an intra-peritoneal injection of 0.1 ml/10g body weight of ketamine (100 mg/ml) and xylazine (20 mg/ml) in sterile saline. For perinatal mice, common anesthesia techniques include the use of isoflurane gas or cryoanesthesia (hypothermia). All anesthesia techniques must be approved by your local IACUC committee before use. We typically use post-natal day (P) 5 mice, although similar techniques can be applied to mice as young as P0 or to adult mice. The surgical procedure cannot begin until the mouse has ceased moving the limbs and no longer responds to toe pinches. For perinatal mice, they will become noticeably lighter in color since the blood flow will slow down, another key to knowing that they are fully under anesthesia.</li>
 <li>In perinatal mice, use Vannas straight scissors to make an approximate 1.5 mm incision along the closed lid fissure. Be careful to cut at the indentation, where the eye will naturally open in the future. This ensures that the lids will heal and open properly during the development of the mouse. A surgical blade can be used for this purpose, although we prefer Vannas scissors since they reduce the risk of puncturing the eye during this procedure. In the video, the scissors were used to directly puncture and cut along the eyelid margin. When learning this technique, forceps should be used to lift the eyelid before cutting and reduce the risk of damaging the eye.</li>
 <li>Pull apart the eyelids with curved dressing forceps to proptose the eye and pinch the eyelids slightly underneath the eye to hold it proptosed for injection. If the lid incision made in step two is too large, the eye will slip back under the lids and prevent adequate exposure during the injection procedure. For adult mice, applying slight pressure around the eye with dressing forceps can hold the eye proptosed for injection.</li>
 <li>Make a small scleral incision at or posterior to the globe's equator by scratching the surface with a 15-degree microsurgery blade. This incision should be only large enough to pass the tip of the injection needle, but no larger than that. If preferred, a small, sharp needle such as an acupuncture needle can be used to make this incision into the subretinal space instead of the microsurgery blade. In darkly pigmented animals, a dark brown-pigmented tissue (the choroid-retinal pigment epithelium) will become visible at the point of incision. In lighter pigmented or albino mice, a surgical marking pen can be applied to the tip of the microsurgery blade to leave an ink spot indicating the incision site. If clear (vitreous) fluid refluxes from the incision site, the blade was placed too deep and entered the vitreous cavity and the therapeutic virus or cells may enter the intravitreal space during injection. For visualization purposes, we have made the incision to this point in the video. It is still possible to complete a subretinal injection in this case, however the retinal detachment caused by the presence of the injection fluid may not fully heal in this mouse. Therefore, care must be taken to cut only the outer surface of the eye and not through the retina before injection. </li>
 <li>Carefully insert the injection needle into the incision site and advance it parallel to the outer eye wall to enter the subretinal space. Advancing too close to the outer wall or too deep within the eye will direct the injection into the wrong location. The best method to understand whether the needle is in the correct location is to practice the technique. Practicing on lightly pigmented or albino mice can help to make precise and accurate injections into the subretinal space. This is because the needle and injection fluid is visible within these eyes.</li>
 <li>Apply light pressure to the syringe plunger and begin injection of the desired fluid. If the needle is correctly placed within the subretinal space, moderate backpressure will be felt. If the needle is within the vitreous, there will be significantly less backpressure and potentially some fluid reflux during the injection. If there is significant backpressure, hold the needle in place for at least an additional 15 seconds before removing the needle slowly. This will allow the high intraocular pressure to equalize instead of refluxing the injected virus or cells back out the incision site. Some of the air and saline may be injected into the subretinal space due to the increased pressure required during the injection procedure. Care should be taken not to over-inject air or saline, since it can flush out the injected virus or cells from the eye. It is also possible to cause permanent retinal detachment from the presence of too much fluid injected into the subretinal space. For visualization purposes, excess dye and air was injected into the subretinal space in the video. It can be clearly seen how some of the dye will be flushed back out of the subretinal space if too much is injected, but also how the air bubble remains within the subretinal space and does not disappear into the vitreous. This was used to emphasize that these injections were all within the correct location of the eye, so one can visualize how the needle should enter the incision site. All injections into the subretinal space cause a temporary retinal detachment due to the presence of the injection fluid. If the injection procedure is completed correctly, this temporary retinal detachment will heal within 24 hr and mice can be used for all further experiments. The eye may remain soft for approximately one week due to the surgical procedure, so a second injection should be avoided during this period of time.</li>
 <li>For neonatal mice, use the curved dressing forceps to gently push the eye back behind the lids and into the orbit. It may be useful to wait for a minute in order to allow the virus to settle within the subretinal space and not cause it to be flushed out of the incision site when gently pushing the eye back into its orbit. After surgery, one drop of 0.5% bupivacaine (marcaine) diluted to 0.25% in sterile saline from a 25-gauge needle can be placed at the injection site. Mice do not show overt signs of distress after subretinal surgery and the use of bupivacaine as a long-acting topical analgesic has been ample for the previous three years. Mice should always be monitored after the surgical procedure to look for any signs of distress; such as infection, swelling, or a decrease in the usual daily activities of the mouse. If these occur, the veterinarian should be notified and further pain medications such as buprenorphine (0.05 mg/kg) can be given as an intraperitoneal injection.</li>
 <li> Wake the mouse from anesthesia according to locally approved animal handling protocol. The mouse should be kept on a heating pad to maintain normal body temperature as it wakes from the anesthetic. The surgical procedure should take less than five minutes to complete, not counting the time for the mouse to undergo anesthesia. Therefore, a heating pad is not necessary during the actual procedure, but may be needed while one is learning the technique as the procedure could take a longer duration of time. The mouse should not be placed back into a clean cage until it has begun to move around on its own. For neonates, gentle toe pinches will help for them to fully recover from the anesthesia, and they should regain their pink color and movement before being placed back with the mother. Mice should be monitored over time for any signs of distress as listed previously.</li>
</ol>

<ol>
	<li>Abe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+retina+using+pigment+epithelial+cell+transplantation.">Regeneration of the retina using pigment epithelial cell transplantation.</a> Nihon Ganka Gakkai Zasshi. 106, 778-803 (2002).</li><li>Jacobson, S. G., Cideciyan, A. V., Ratnakaram, R., Heon, E., Schwartz, S. B., Roman, A. J., Peden, M. C., Aleman, T. S., Boye, S. L., Sumaroka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+Leber+congenital+amaurosis+caused+by+RPE65+mutations:+safety+and+efficacy+in+15+children+and+adults+followed+up+to+3+years.">Gene therapy for Leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years.</a> Arch. Ophthalmol. 130, 9-24 (2011).</li><li>Schwartz, S. D., Hubschman, J. P., Heilwell, G., Franco-Cardenas, V., Pan, C. K., Ostrick, R. M., Mickunas, E., Gay, R., Klimanskaya, I., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379, 713-720 (2012).</li><li>Wang, N. K., Tosi, J., Kasanuki, J. M., Chou, C. L., Kong, J., Parmalee, N., Wert, K. J., Allikmets, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+reprogrammed+embryonic+stem+cells+improves+visual+function+in+a+mouse+model+for+retinitis+pigmentosa.">Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.</a> Transplantation. 89, 911-919 (2010).</li><li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Tosi, J., Sancho-Pelluz, J., Davis, R. J., Hsu, C. W., Wolpert, K. V., Sengillo, J. D., Lin, C. S., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-mediated+expression+of+cDNA+and+shRNA+slows+degeneration+in+retinitis+pigmentosa.">Lentivirus-mediated expression of cDNA and shRNA slows degeneration in retinitis pigmentosa.</a> Exp. Biol. Med. 236, 1211-1217 (2011).</li><li>Tucker, B. A., Park, I. H., Qi, S. D., Klassen, H. J., Jiang, C., Yao, J., Redenti, S., Daley, G. Q., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+adult+mouse+iPS+cell-derived+photoreceptor+precursors+restores+retinal+structure+and+function+in+degenerative+mice.">Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.</a> PLoS One. 6, e189992 (2011).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A high-throughput Mouse Eye Phenomics System. , (2010).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384 (2011).</li></ol>

No conflicts of interest declared.

This video technique provides instructions on completing the subretinal injection surgical procedure successfully, and ensuring that the gene therapy vector or stem cells are placed in the location necessary to efficiently treat the ophthalmic disease. This technique allows for the targeting of retinal cells such as the RPE or photoreceptors, since it places the gene therapy vectors or stem cell-derived tissues in the vicinity of these cells. Previous methods involved intravitreal injections, where the fluid is placed wi...

<table><tbody><tr><td>0.8-1.10 x 100 mm Capillary Tube (glass)</td><td>Kimble Glass, Inc.</td><td>34502 99</td><td></td></tr><tr><td>Flaming/Brown Micropipette Puller </td><td>Sutter Instrument</td><td>P-97</td><td>Narishige microforge can be used instead. Catalog #MF-900 </td></tr><tr><td>Sigmacote</td><td>Sigma Aldrich</td><td>SL2-25ML</td><td>Silicone</td></tr><tr><td>Dubecco&#039;s Phosphate Buffered Saline with Calcium Chloride and Magnesium Chloride</td><td>Gibco-Invitrogen</td><td>14040-133</td><td></td></tr><tr><td>Safety-Lok 25 3/4G x 12&quot;; Blood Collection Set</td><td>B-D Vacutainer</td><td>367298</td><td></td></tr><tr><td>1 ml Sub-Q 26 5/8G Slip-Tip Syringe</td><td>Becton-Dickinson</td><td>309597</td><td></td></tr><tr><td>0.5-10 &amp;mu;l Finnpipette II Adjustable-Volume Pipetter</td><td>Fisherbrand</td><td>21-377-815</td><td></td></tr><tr><td>1-200 &amp;mu;l Natural Beveled Tips</td><td>USA Scientific, Inc.</td><td>1111-1700</td><td></td></tr><tr><td>Discovery.V8 Stereo Microscope</td><td>Zeiss</td><td>MC1500</td><td></td></tr><tr><td>60 mm x 15 mm Style Treated Polystyrene Cell Culture Dish</td><td>Corning Incorporated</td><td>430166</td><td></td></tr><tr><td>Vannas Straight Scissors</td><td>Storz Ophthalmics</td><td>E3383 S</td><td></td></tr><tr><td>Curved Dressing Forceps with Serrations Delicate</td><td>Storz Ophthalmics</td><td>E1408</td><td></td></tr><tr><td>15 Degree Microsurgery Knife</td><td>Wilson Ophthalmic Corp.</td><td>091204</td><td></td></tr><tr><td>Ketamine</td><td>Ketaset III</td><td>NADA #45-290</td><td></td></tr><tr><td>Xylazine</td><td>Lloyd Laboratories</td><td>NADA #139-236</td><td></td></tr><tr><td>Bupivacaine (Marcaine)</td><td>AstraZeneca</td><td>N/A</td><td></td></tr><tr><td>Buprenorphine </td><td>Sigma Aldrich</td><td>B9275</td><td></td></tr></tbody></table>

subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

A drawing of the mouse eye is shown with major structures labeled for reference, with arrows displaying the locations for both intravitreal and subretinal injection surgical procedures (arrowheads, Figure 1). Gene therapy vectors, such as the lacZ lentiviral vector (Figure 2), can be injected using these locations. Additionally, stem cells, such as mouse embryonic stem cells (Figure 3), can also be transplanted at these sites in the mouse eye. 
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Watch this Scientific Journal Video about Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye at JoVE.com

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

subretinal-injection-gene-therapy-vectors-stem-cells-perinatal-mouse

Research

JoVE Journal

Medicine

33.1K Views.  Columbia University. This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

Video: Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Neuroscience

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Biology

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

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

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

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