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

Summary

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

Abstract

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.

Protocol

1. Assemble Devices for the Subretinal Injection

1. Purchase or make a 100 μm 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 μm). 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....

Results

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.

Discussion

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.......

Materials

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