title: Optimizing gene transfer to conventional outflow cells in living mouse eyes.
abstract: The mouse eye has physiological and genetic advantages to study conventional outflow function. However, its small size and shallow anterior chamber presents technical challenges to efficient intracameral delivery of genetic material to conventional outflow cells. The goal of this study was to optimize methods to overcome this technical hurdle, without damaging ocular structures or compromising outflow function. Gene targeting was monitored by immunofluorescence microscopy after transduction of adenovirus encoding green fluorescent protein driven by a CMV promoter. Guided by a micromanipulator and stereomicroscope, virus was delivered intracamerally to anesthetized mice by bolus injection using a 33 gauge needle attached to Hamilton syringe or infusion with glass micropipette connected to syringe pump. The total number of particles introduced remained constant, while volume of injected virus solution (3-10 μl) was varied for each method and time of infusion (3-40 min) tested. Outflow facility and intraocular pressure were monitored invasively using established techniques. Unlike bolus injections or slow infusions, introduction of virus intracamerally during rapid infusions (3 min) at any volume tested preferentially targeted trabecular meshwork and Schlemm's canal cells, with minimal transduction of neighboring cells. While infusions resulted in transient intraocular pressure spikes (commensurate with volume infused, Δ40-70 mmHg), eyes typically recovered within 60 min. Transduced eyes displayed normal outflow facility and tissue morphology 3-6 days after infusions. Taken together, fast infusion of virus solution in small volumes intracamerally is a novel and effective method to selectively deliver agents to conventional outflow cells in living mice.
authors: Department of Ophthalmology, Duke University School of Medicine, Box 3802, Durham, NC 27710, USA.
authors: G
authors: G
authors: Li
authors: P
authors: P
authors: Gonzalez
authors: L J
authors: LJ
authors: Camras
authors: I
authors: I
authors: Navarro
authors: J
authors: J
authors: Qiu
authors: P
authors: P
authors: Challa
authors: W D
authors: WD
authors: Stamer
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000869:Anterior Eye Segment / Q000378:metabolism; D001082:Aqueous Humor / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D005123:Eye / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005901:Glaucoma / Q000628:therapy; D049452:Green Fluorescent Proteins / Q000235:genetics*; D056965:Injections, Intraocular / Q000009:adverse effects / Q000295:instrumentation / Q000379:methods*; D007429:Intraocular Pressure; D051379:Mice; D008810:Mice, Inbred C57BL; D008845:Microinjections / Q000009:adverse effects / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics; D014129:Trabecular Meshwork / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins