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Bogale Aredo, Yi Ding, Xin Zhong, Cynthia Xin-Zhao Wang, Bruce Beutler, Rafael Ufret-Vincenty; Forward genetics: searching for novel genes essential to retinal development and function. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1841. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Our aim is to identify novel genes associated to retinal development and function, using a novel and powerful forward genetics approach. This approach relies on generating random mutations and identifying mice expressing strong phenotypes in order to establish gene-phenotype associations.
A large number of mice with random mutations were generated in collaboration with Dr. Bruce Beutler’s laboratory, using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis and a special breeding protocol. Whole exome sequencing of G1 progenitors allows for the identification of all possible mutations. Then their zygosity is established in G2/G3 mice before phenotypic assessment. We examine mice at around 6 months of age by fundus photography and OCT using a Micron IV system (Phoenix Research Laboratories, Pleasanton, CA). Our findings are then analyzed by a powerful software (Linkage Analyzer) that allows us to establish gene-phenotype associations almost immediately after phenotyping. Parameters that were analyzed included: total retinal thickness, outer retinal thickness, outer nuclear layer thickness, yellow fundus spots, chorioretinal lesions, retinal vasculature anomalies and retinal detachment.
We will present data from our results having examined over 1000 mice. Preliminary results showed 27 gene-phenotype associations in the initial 600 mice. Strong gene-phenotype associations were identified for all the quantitative OCT parameters. Associations were also identified for some of the qualitative parameters, including chorioretinal lesions and yellow fundus spots. Some of the identified gene-phenotype associations have been described in the literature, providing confirmation of the validity of our approach.
This forward genetics approach is allowing us to identify novel genes associated with retinal development, function and disease. In the future, we plan to further characterize the effects of some of the most interesting genes by generating Crispr/Cas9 mutated mice. We also hope to use these techniques to identify genes important in the response to retinal oxidative stress in an unbiased manner.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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