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P. Lagali, D. Balya, D. Kim, C. Cepko, B. Roska; Targeted Reporter Gene Expression for Morphological and Functional Assessment of Inner Retinal Neurons in Wild-Type and Retinal Degeneration Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5944.
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© ARVO (1962-2015); The Authors (2016-present)
To deliver functional reporter constructs into the inner retinas of wild-type and retinal degeneration mice in order to study the structure, function, and circuitry of inner nuclear layer retinal neurons in normal and diseased states.
Functional fluorescent reporter protein-encoding DNA constructs were engineered and delivered to mouse inner retinal cells using in vivo electroporation, viral transduction, and the creation of transgenic mice. Various regulatory elements were tested for optimal expression of the reporter proteins in the cell types of interest. Retinal gene expression was analyzed by immunohistochemistry. Reporter gene function in retinal neurons was assessed using electrophysiological methods.
Reporter gene expression was restricted to specific subsets of inner retinal neurons depending on a combination of the method of gene delivery and the choice of genetic regulatory elements employed. Reporter gene expression in specific amacrine and bipolar cell types was observed based on morphological characterization of the fluorescently labeled cells and on cell type-specific marker protein localization. Physiological responses to visual stimuli were recorded from retinal ganglion cells that were synaptically connected to the labeled inner nuclear neurons in both wild-type and retinal degeneration mice. Expression of a reporter gene encoding a photosensory protein within inner retinal cells resulted in the generation of electrical responses in ganglion cells upon presentation of photic stimuli.
Gene expression targeted to specific subtypes of cells within the inner retina can be achieved using various gene delivery methods coupled with sequence elements directing cell type-restricted transcription. Functional analysis of targeted cells enables characterization of the neuronal circuitry of specific inner retinal cell types. Our results further reveal that visual processing capabilities are supported by the inner retinas of mice with retinal degeneration.
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