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Rafael Ufret-Vincenty, Bogale Aredo, Bo Chen, Yuanfei Zhu, Yi Ding, Cynthia X Zhao, Bruce Beutler; Forward genetics: searching for novel genes essential to retinal development and homeostasis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6060. doi: https://doi.org/.
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Our aim is to identify novel genes associated to retinal development and homeostasis using a state-of-the-art forward genetics approach. This approach relies on generating random mutations and identifying mice expressing retinal 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, and yellow fundus spots.
Looking at fundus photographs and OCT, we examined 3871 G3 mice, with null or potentially damaging mutations covering an estimated 11.4% of the genome. From these we identified 53 genes with a statistically significant association with retinal anatomy. As a validation of our approach, six of the identified genes (Cngb1, Tmem135, Impdh1, Poc5, Cacna2d4 and Cnga1) have been shown to cause forms of retinal dystrophy/degeneration. Also, a gene that we found to be associated to subretinal lesions (Vldlr) is known to be associated to the development of choroidal/retinal neovascularization.
This forward genetics approach is allowing us to identify novel genes associated with retinal development, function and disease. The biggest advantage of our approach compared to others, is that all G3 mice used in screening have been pre-genotyped at all mutant loci. In the future, we plan to further characterize the effects of some of the most interesting genes we have identified 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 abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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