Molecular VisionPub Date : 2025-01-12eCollection Date: 2025-01-01
Fangyu Lin, Su-Ting Lin, Jiaxing Wang, Jana T Sellers, Micah A Chrenek, John M Nickerson, Jeffrey H Boatright, Eldon E Geisert
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An improved method of transducing retinal ganglion cells using AAV via transpupillary injection in adult mouse eyes.
Purpose: Intravitreal injection of adeno-associated virus (AAV) vectors is a good approach for transducing retinal ganglion cells (RGCs) in mice. It allows for high transduction efficiency and is relatively specific to RGCs. To deliver vectors, most studies use a transscleral approach that can have potentially negative effects, causing damage to the lens or retina. We optimized the intravitreal injection method using a transpupillary approach to minimize ocular damage and efficiently transfect RGCs.
Methods: C57BL/6J mice were anesthetized, and their irises were dilated. The eyeball was held with forceps while a small, full-thickness incision was made halfway between the center and periphery of the cornea. Using a bent 35-gauge blunt needle, the tip was navigated through the incision across the anterior chamber to reach the distal aspect of the pupil. The needle was inserted through the pupil, swept around the lens, and entered the vitreous, delivering expression vectors containing cytomegalovirus (CMV) promoter-driving green fluorescent protein (AAV-CMV-GFP) into the vitreous chamber. Fourteen days after injection, live fluorescent fundus images were taken, followed by immunostaining for GFP.
Results: With the improved injection technique, the lens remained clear and undamaged. Fundus imaging and GFP staining showed that over 90% of the mouse retinas sustained no visible damage. Retinas injected via the transpupillary approach also exhibited GFP transduction throughout the ganglion cell layer.
Conclusions: Transpupillary intravitreal injection reduces the potential risk compared to the transscleral approach, offering a promising and efficient method for delivering reporter genes to RGCs and ensuring high levels of gene expression without damage to the lens or retina.
期刊介绍：
Molecular Vision is a peer-reviewed journal dedicated to the dissemination of research results in molecular biology, cell biology, and the genetics of the visual system (ocular and cortical).
Molecular Vision publishes articles presenting original research that has not previously been published and comprehensive articles reviewing the current status of a particular field or topic. Submissions to Molecular Vision are subjected to rigorous peer review. Molecular Vision does NOT publish preprints.
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