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

Abstract

Source: Park, S. W., et al. Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium. J. Vis. Exp. (2015)

The video describes the method for subretinal injection of viral vectors into the retinal pigment epithelium (RPE) in a mouse model. It details the procedure from anesthesia and eye preparation to precise lentiviral vector delivery, ensuring gene integration and expression in the RPE.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparing Injection Kit and Viral Vectors

1. Prepare the microliter syringe equipped with a 33 G blunt needle sterilized using ethylene oxide gas. Dilute the viral vectors in PBS (Phosphate buffered saline) for adequate titer in the microtube (i.e., 1 x 10⁶ TU/µl). Flush the syringe several times with viral vectors to remove any dead space in the syringe.

2. Subretinal Injection of Viral Vectors

1. Anesthetize the adult mice (i.e., 6 - 8 weeks old) with an intraperitoneal injectio....

Results

Figure 1. Schematic Diagram of the Subretinal Injection. H&E (Hematoxylin and Eosin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tweezers DUMONT #5 11cm DUMOSTAR 0.1 x 0.06 mm tips	WPI	500233
VANNAS Scissors S/S, 105mm	WPI	555583S
33G Blunt needle	WPI	NF33BL-2
NanoFil Syringe, 10 microliter	WPI	NANOFIL
RPE-KIT	WPI	RPE-KIT	For easy one hand injection
30Gx1/2 (0.3mmx 13mm) BD PrecisionGlideTM Needle	BD	305107	Initial puncture for subretinal injection
Microscope Cover Glasses (No. 1 3 mm diameter)	Warner Instruments	64-0720 (CS-3R)
Leica operating microscope	Leica	LM M80
Fluoresecein microscope	Nikon	Eclipse 80i
Lentivirus	Thermo scientific	TMO.LV-Ctr	Used to dilute vectors
PBS	Gibco	10010-015	Used to dilute vectors
Troperin (Phenylephrin 0.5%-Tropicamide 0.5%)	Hanmi		For dilation
Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% (Sterile)	Bausch&Lomb		For topical anesthesia
Healon GV OVD	Abbott Medical Optics Inc.
Zoletil 50 (tiletamine hypochloride and zolazepam hypochloride)	Virbac		For general anesthesia
Rompun® injection (Xylazine HCl)	Bayer		For general anesthesia