An Avian Adeno-Associated Viral Vector for Visualization of Post-Natal Chick Retinal Circuitry

Abstract

Purpose : The post-hatching chicken is a valuable animal model for research, but the molecular tools needed for altering its gene expression are not yet available. Our purpose here was to adapt the adeno-associated viral (AAV) vector method, used widely in mammalian studies, for use in investigations of the chicken retina. We hypothesized that the recently characterized avian adeno-associated viral (A3V) vector could effectively transduce chick retinal neurons for in situ visualization, morphological characterization, and ultimately, genetic manipulation.

Methods : A3V encoding green fluorescent protein (A3V-GFP) was produced via the triple-plasmid transfection method and iodixanol gradient purification. Concentrated A3V-GFP (10¹¹-10¹² genome copies/mL) was co-injected with varying concentrations of the glycosidic enzymes heparinase III (E.C. 4.2.2.8) and hyaluronan lyase (E.C. 4.2.2.1) into P1-3 chick vitreous humour and incubated for 7-10 days. Whole retinas were then flat-mounted and visualized via laser-scanning confocal microscopy for analysis of expression and imaging of individual retinal cells. ImageJ v2.0.0, Simple Neurite Tracer v3.0.5, and Volume Viewer v2.01 were used for imaging and reconstruction of retinal cells.

Results : Intravitreal A3V-GFP injection resulted in GFP expression in a small percent of retinal cells, primarily those with processes and/or cell bodies near the vitreal surface (ganglion cells, Müller cells). Co-injection of glycosidic enzymes increased the depth and breadth of viral transduction, allowing for visualization of more outer retinal neurons. Reconstruction of isolated cells allowed for computational analysis and 3D visualization of morphology.

Conclusions : Intravitreal A3V injection is a promising method for investigating chick retinal cells and circuitry in situ. The use of viral vectors uniquely allows for the use of cell-specific promoters, which may be used to unravel morphologies of specific marker expressing cell-types. Future developments to increase transduction efficiency of A3V may allow for its use in gene knock-in and knockout experiments, thus vastly increasing the experimental potential of this valuable animal model.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Retinal cells expressing GFP following transduction with A3V-GFP, including a retinal ganglion cell (left, compressed Z-stack), horizontal cell (middle, compressed Z-stack) and Müller glial cell (right, 3D reconstruction). Scale bars = 25 μm.