Author

Jacob Fleming

Document Type

Honors Capstone Project

Date of Submission

Spring 5-1-2008

Capstone Advisor

Dr. Michael Zuber

Honors Reader

Dr. Craig Albertson

Capstone Major

Biology

Capstone College

Arts and Science

Audio/Visual Component

no

Capstone Prize Winner

no

Won Capstone Funding

no

Honors Categories

Sciences and Engineering

Subject Categories

Biology

Abstract

Currently, 3.5 million Americans over the age of 40 are blind or visually impaired. Cases of visual impairment can be attributed to a number of causes. Retinitis pigmentosa, glaucoma, and macular degeneration all involve some form of damage to the retina. While medical advances continue to aid those with vision loss, there is currently no cure for visual impairment due to cell death. In the age of stem cells however, there is hope for those with retinal damage.

Seven eye field transcription factors play specific roles in eye development. When injected into developing Xenopus laevis, these transcription factors induce the formation of an ectopic eye field and eventually eyes outside of the nervous system. These findings demonstrate that induced retinal stem cells can differentiate and form a functional retina in a developing embryo. However, for these cells to heal the damaged retina, they must also functionally replace dead or dying cells in a mature retina. In order to do this, an assay has to be developed that will allow us to test visual function.

Two behavioral assays developed to assess visual function of Xenopus laevis were considered. The OptoMotry system and a second phototropic behavioral assay using color preference as its basis were considered. After various attempts to modify the background color preference assay, a successful design was developed utilizing Xenopus characteristic behavioral response.

The bacterial protein nitroreductase converts the relatively nontoxic prodrug CB1954 to a cytotoxic alkylating agent. This protein was used to create a transgenic line of Xenopus laevis that drives nitroretuctase (NR) expression under the control of the rhodopsin promoter. In the mature retina, expression from the Xenopus rhodopsin promoter (XOP) is restricted to the rod photoreceptors. Rod cells, one of two photoreceptor types in the retina (cones are the other), are responsible for vision at low light levels. Additionally, rod cells are generally the first to be degraded when visual loss occurs. When the nitroreductase transgenic line (XOP-NR) is treated with CB 1954, rod death occurs.

The behavioral assay operates under varying levels of light to exploit our ability to selectively ablate rod photoreceptors. If transgenic tadpoles with rod degeneration show a different behavioral response than wild types at low light levels, then this assay can be used to quantify retinal degeneration.

This assay has been successfully used to quantitatively show variance in response of wild types in relation to the quantity of ambient light. The response curve for XOP-NR animals using this assay was delayed for a number of reasons. The current method for producing the transgenic line of Xenopus provides approximately 40% XOP-NR animals and 60% wild types. This alone provides the difficult task of sorting out which animals are transgenic. The future of this assay relies on genotyping live animals before testing.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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