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Fig. 8 The EGFP-tagged zebrafish rlbp1b gene but not the human RLBP1 gene can rescue the dim light OKR in rlbp1b knockout larvae. (A) Schematic of standard raising and dim light OKR approach used to assess rescue in humanized zebrafish lines. (B) Schematic of expression vectors used to create humanized EGFP-tagged RLBP1 zebrafish lines used in this study. (C) The EGFP-tagged human RLBP1 gene is not sufficient to rescue the dim light OKR phenotype in rlbp1b knockout larvae (p < 0.0001, Mann Whitney test). Three biological replicates n ≥ 16 larvae per group. (D, E) The expression of missense RLBP1 disease variants p.R151Q and p.A237V do not cause any additional impairment of the dim light OKR response in rlbp1b knockout larvae. p.R151Q 3 biological replicates n ≥ 12 larvae per group (p < 0.0001, two tailed t-test). p.A237V 3 biological replicates n ≥ 11 larvae per group (p < 0.0047, Welch's t-test). (F) Schematic of expression vectors used to create zebrafish lines expressing EGFP-tagged rlbp1b used in this study. (G) F0 rescue of the dim light OKR phenotype using the tg(rpe65a:eGFP-rlbp1b) transgene in the rlbp1b knockout larvae. Three biological replicates n = 24 rlbp1b−/− larvae per group (p = 0.0086, two tailed t-test). (H) Rescue of the dim light OKR phenotype in F1 larvae stably expressing tg(rpe65a:eGFP-rlbp1b). Three biological replicates n = 27 rlbp1b−/− larvae per group (p < 0.0001, two tailed t-test). (I) Stable expression of the tg(rpe65a:eGFP-rlbp1bp.R151Q) fails to rescue the dim light OKR phenotype in rlbp1b knockout larvae. No significant difference between non-transgenic larvae and larvae with the tg(rpe65a:eGFP-rlbp1bp.R151Q) transgene. Significant difference between larvae with the wildtype vs. p.R151Q mutant transgene (p < 0.0053, Kruskal–Wallis test). Three biological replicates n = 25 rlbp1b−/− larvae per group. All graphs show mean ± standard deviation.