Fig. 1: Experimental protocol for forward genetics screening, identification of gene-phenotype associations to fundus spot accumulation and characterization of phenotypes.

Mutagenesis was induced with N-ethyl-N-nitrosourea (ENU) (a). A special breeding protocol is used to ensure that we include potential dominant and recessive mutations (b). G3 mice were screened using a fundus spot semiquantitative scale (c). Statistically significant gene-phenotype associations were identified using special software (d). Genes of interest were targeted using CRISPR/Cas9 technology (e) and the resulting mice were phenotyped (morphology, ultrastructure, physiology, biochemistry) with a variety of techniques (f). Using this protocol, the reservoir allele was identified, and a Manhattan Plot (g) and scatter plot (h) show its statistically significant association to fundus spot accumulation. No significant association between the reservoir allele and retinal layer thickness was detected in this pedigree for any of our OCT parameters (i). The scatter plots (h, i) show individual mice plus the error bars (Mean ± Standard Deviation). Figure 1a–f was generated with BioRender.com.